Processing biomass

ABSTRACT

Biomass (e.g., plant biomass, animal biomass, and municipal waste biomass) is processed for use in the production of useful products, such as fuels. For example, systems can use biomass materials, such as cellulosic and/or lignocellulosic materials, to enhance the production of a product, e.g., the production of ethanol and/or butanol by fermentation.

RELATED APPLICATIONS

This application claims priority to copending U.S. application Ser. No.13/662,763 filed Oct. 29, 2013; which claims priority to U.S.application Ser. No. 12/782,543 filed May 18, 2010, U.S. Pat. No.8,377,668 issued Feb. 19, 2013; which claimed priority to U.S.Provisional Application Ser. No. 61/180,032, filed May 20, 2009, andU.S. Provisional Application Ser. No. 61/252,293, filed Oct. 16, 2009.The complete disclosure of each of these is hereby incorporated byreference herein.

BACKGROUND

Cellulosic and lignocellulosic materials are produced, processed, andused in large quantities in a number of applications. Often suchmaterials are used once, and then discarded as waste, or are simplyconsidered to be waste materials, e.g., sewage, bagasse, sawdust, andstover.

Various cellulosic and lignocellulosic materials, their uses, andapplications have been described in U.S. Pat. Nos. 7,307,108, 7,074,918,6,448,307, 6,258,876, 6,207,729, 5,973,035 and 5,952,105; and in variouspatent applications, including “FIBROUS MATERIALS AND COMPOSITES,”PCT/US2006/010648, filed on Mar. 23, 2006, and “FIBROUS MATERIALS ANDCOMPOSITES,” U.S. Patent Application Publication No. 2007/0045456.

SUMMARY

In some instances, the presence of biomass in a process, for examplefermentation, facilitates conversion of a low molecular weight sugar toan intermediate or a product. The inventors have found that includingbiomass in a mixture with a low molecular weight sugar, a medium, e.g.,a solvent or solvent system, and a microorganism can improve the yieldand production rate of an intermediate or a product obtained byconversion of the sugar, for example an alcohol such as ethanol orbutanol. Including the biomass can also prevent incomplete, sluggish, or“stuck” product conversion, e.g., by fermentation.

The biomass may not in itself be converted to the product (such as analcohol), or may be partially or fully converted to the product alongwith the low molecular weight sugar.

In instances where the biomass is partially converted, the surface areaand porosity of the biomass is increased relative to the surface areaand porosity of the starting biomass, which can advantageously increasethe conversion rate of the low molecular weight sugar to the product.

In some cases, the biomass may be the remnants of a cellulosic orlignocellulosic material that has been saccharified, e.g., lignin and/orother materials that are left over after cellulose has been converted tosugar.

In one aspect, the invention features a method that includes utilizing amicroorganism and/or enzyme that is immobilized on a biomass material,e.g., functionalized biomass fibers, to convert a carbohydrate, e.g., alow molecular weight sugar, to a product. By “immobilized,” it is meantthat the microorganism and/or enzyme is bonded, directly or indirectly(e.g., through a chemical linker), to the fibers by covalent, hydrogen,ionic, or equivalent bonding, and/or by mechanical interaction, e.g.,between the microorganism and pores of the biomass material, e.g.,fibers. Bonding may be created, e.g., by electrically polarizing thebiomass material. The interaction can be permanent, semi-permanent, orfleeting. Mechanical interaction may include the microorganism or enzymenesting in or clinging to pores or other sites of the biomass material.

Some implementations include one or more of the following features.

Converting can include allowing the microorganism to convert at least aportion of the low molecular weight sugar to an alcohol, e.g., ethanolor butanol, or to a hydrocarbon or hydrogen. Converting may includefermentation. The microorganism may comprise a yeast, e.g., S.cerevisiae and/or P. stipitis, or a bacterium, e.g., Zymomonas mobilis.The method may further include irradiating the biomass fibers, e.g.,with ionizing radiation, for example using a particle beam. The biomassfibers may have a BET surface area of greater than 0.25 m²/g, and/or aporosity of at least 70%. The biomass fibers may be derived from abiomass material that has internal fibers, and that has been sheared toan extent that its internal fibers are substantially exposed.

In another aspect, the invention features a mixture that includes abiomass material having polar functional groups, a microorganism havingcomplementary attractive functional groups, and a liquid medium.

In a further aspect, the invention features a composition comprisingbiomass fibers having functional groups, and a microorganism havingcomplementary attractive functional groups, the microorganism beingimmobilized on the biomass fibers.

The invention also features a method that includes converting a lowmolecular weight sugar, or a material that includes a low molecularweight sugar, in a mixture with a biomass, a microorganism, and asolvent or a solvent system, e.g., water or a mixture of water and anorganic solvent, to a product. Examples of solvents or solvent systemsinclude water, hexane, hexadecane, glycerol, chloroform, toluene, ethylacetate, petroleum ether, liquefied petroleum gas (LPG), ionic liquidsand mixtures thereof. The solvent or solvent system can be in the formof a single phase or two or more phases. The biomass can be, e.g., infibrous form.

In some instances, having a biomass material (e.g., treated by anymethod described herein or untreated) present during production of aproduct, can enhance the production rate of the product. Without wishingto be bound by any particular theory, it is believed that having a solidpresent, such as a high surface area and/or high porosity solid, canincrease reaction rates by increasing the effective concentration ofsolutes and providing a substrate on which reactions can occur.

In some embodiments, a biomass material that has been irradiated,oxidized, chemically treated, mechanically treated, sonicated, steamexploded and/or pyrolyzed, can be added to a low molecular weight sugarfermentation process, e.g., to enhance fermentation rate and output.

For example, an irradiated or an un-irradiated biomass material, e.g., apaper fiber, can be added to a fermentation process, such as during acorn-ethanol fermentation or a sugarcane extract fermentation, toincrease the rate of production by at least 10, 15, 20, 30, 40, 50, 75,100 percent or more, e.g., at least 150 percent, or even up to 1000percent. Conversion, e.g., fermentation, can exhibit a percentperformance, as defined in the Examples herein, of at least 140%, insome cases at least 170%.

The biomass material can have a high surface area, high porosity, and/orlow bulk density. In some embodiments, the biomass is present in themixture from about 0.5 percent to about 50 percent by weight, such asbetween about 1 percent and about 25 percent by weight, or between about2 percent and about 12.5 percent by weight. In other embodiments, thebiomass is present in amounts greater than about 0.5 percent by weight,such as greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or even greaterthan about 10 percent by weight.

Because the biomass material is not itself consumed during theconversion process, the biomass material can be reused in multiple batchprocesses, or can be used continuously for the production of arelatively large volume of the product.

Some implementations include one or more of the following features. Themethod can include irradiating the fibrous biomass prior to mixing,e.g., with ionizing radiation, for example at a total dosage of at least5 Mrad. Irradiating can be performed using a particle beam. Irradiatingcan be conducted under conditions selected to reduce the molecularweight of the biomass. Irradiation can be performed using multipleapplications of radiation. The ionizing radiation can include electronbeam radiation. For example, the radiation can be applied at a totaldose of between about 10 Mrad and about 150 Mrad, such as at a dose rateof about 0.5 to about 10 Mrad/day, or 1 Mrad/s to about 10 Mrad/s. Insome embodiments, irradiating includes applying two or more radiationsources, such as gamma rays and a beam of electrons.

In some embodiments, irradiating is performed on the biomass feedstockwhile the biomass feedstock is exposed to air, nitrogen, oxygen, helium,or argon. In some embodiments, pretreatment can include pretreating thebiomass feedstock with steam explosion.

In some embodiments, the method includes mechanically treating thebiomass, e.g., by reducing one or more dimensions of individual piecesof biomass, for example by shearing, stone grinding, mechanicallyripping or tearing, pin grinding, wet or dry grinding, air attritionmilling, cutting, squeezing, compressing or combinations of any of theseprocesses. In some cases, after mechanical treatment the biomassincludes fibers having an average length-to-diameter ratio of greaterthan 5/1. In some embodiments, the prepared biomass can have a BETsurface area of greater than 0.25 m²/g. The mechanically treated biomasscan have a bulk density of less than about 0.5 g/cm³, e.g., less than0.35 g/cm³.

In any of the methods disclosed herein, radiation may be applied from adevice that is in a vault.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram illustrating treatment of biomass and the useof the treated biomass in a fermentation process.

FIG. 2 is a schematic representation of functionalized biomassinteracting with a microorganism.

FIG. 3 is an infrared spectrum of Kraft board paper sheared on a rotaryknife cutter.

FIG. 4 is an infrared spectrum of the Kraft paper of FIG. 3 afterirradiation with 100 Mrad of gamma radiation.

FIGS. 5A-5I are ¹H-NMR spectra of samples P132, P132-10, P132-100, P-1e,P-5e, P-10e, P-30e, P-70e, and P-100e in Example 13. FIG. 5J is acomparison of the exchangeable proton at ˜16 ppm from FIGS. 5A-5I. FIG.5K is a ¹³C-NMR of sample P-100e. FIGS. 5L-5M are ¹³C-NMR of sampleP-100e with a delay time of 10 seconds. FIG. 5N is a ¹H-NMR at aconcentration of 10% wt./wt. of sample P-100e.

FIG. 6 is a graph showing glucose concentration over time.

FIG. 7 is a graph showing cell concentrations for Z. mobilis.

FIG. 8 is a graph showing ethanaol concentrations obtained utilizing Z.mobilis.

FIG. 9 is a graph showing the percentage of growth and ethanolproduction obtained utilizing Z. mobilis.

FIG. 10 is a graph showing cell concentrations for P. stipitis.

FIG. 11 is a graph showing ethanaol concentrations obtained utilizing P.stipitis.

FIG. 12 is a graph showing the percentage of growth and ethanolproduction obtained utilizing P. stipitis.

FIG. 13 is a graph showing cell concentrations for S. cerevisiae.

FIG. 14 is a graph showing ethanol concentrations obtained utilizing S.cerevisiae.

FIG. 15 is a graph showing the percentage of growth and ethanolproduction obtained utilizing S. cerevisiae.

DETAILED DESCRIPTION

Functionalized biomass materials having desired types and amounts offunctionality, such as carboxylic acid groups, enol groups, aldehydegroups, ketone groups, nitrile groups, nitro groups, or nitroso groups,can be prepared using the methods described herein. Such functionalizedmaterials can facilitate conversion of low molecular weight sugar to aproduct, e.g., during a fermentation process.

Types of Biomass

Preferred biomass materials for use in the processes described hereincontain fibers which can be functionalized with functional groups thatare complementary with functional groups on an agent to be used inconverting the sugar, e.g., a microorganism such as yeast.

Fiber sources include cellulosic fiber sources, including paper andpaper products (e.g., polycoated paper and Kraft paper), andlignocellulosic fiber sources, including wood, and wood-relatedmaterials, e.g., particleboard. Other suitable fiber sources includenatural fiber sources, e.g., grasses, rice hulls, bagasse, jute, hemp,flax, bamboo, sisal, abaca, straw, switchgrass, alfalfa, hay, corn cobs,corn stover, coconut hair; fiber sources high in α-cellulose content,e.g., cotton; and synthetic fiber sources, e.g., extruded yarn (orientedyarn or un-oriented yarn). Natural or synthetic fiber sources can beobtained from virgin scrap textile materials, e.g., remnants or they canbe post consumer waste, e.g., rags. When paper products are used asfiber sources, they can be virgin materials, e.g., scrap virginmaterials, or they can be post-consumer waste. Aside from virgin rawmaterials, post-consumer, industrial (e.g., offal), and processing waste(e.g., effluent from paper processing) can also be used as fibersources. Also, the fiber source can be obtained or derived from human(e.g., sewage), animal or plant wastes. Additional fiber sources havebeen described in U.S. Pat. Nos. 6,448,307, 6,258,876, 6,207,729,5,973,035 and 5,952,105.

In some embodiments, the biomass material includes a carbohydrate thatis or includes a material having one or more β-1,4-linkages and having anumber average molecular weight between about 3,000 and 50,000. Such acarbohydrate is or includes cellulose (I), which is derived from(β-glucose 1) through condensation of β(1,4)-glycosidic bonds. Thislinkage contrasts itself with that for α(1,4)-glycosidic bonds presentin starch and other carbohydrates.

Starchy materials include starch itself, e.g., corn starch, wheatstarch, potato starch or rice starch, a derivative of starch, or amaterial that includes starch, such as an edible food product or a crop.For example, the starchy material can be arracacha, buckwheat, banana,barley, cassava, kudzu, oca, sago, sorghum, regular household potatoes,sweet potato, taro, yams, or one or more beans, such as favas, lentilsor peas. Blends of any two or more starchy materials are also starchymaterials.

In some cases the biomass is a microbial material. Microbial sourcesinclude, but are not limited to, any naturally occurring or geneticallymodified microorganism or organism that contains or is capable ofproviding a source of carbohydrates (e.g., cellulose), for example,protists, e.g., animal protists (e.g., protozoa such as flagellates,amoeboids, ciliates, and sporozoa) and plant protists (e.g., algae suchalveolates, chlorarachniophytes, cryptomonads, euglenids, glaucophytes,haptophytes, red algae, stramenopiles, and viridaeplantae). Otherexamples include seaweed, plankton (e.g., macroplankton, mesoplankton,microplankton, nanoplankton, picoplankton, and femptoplankton),phytoplankton, bacteria (e.g., gram positive bacteria, gram negativebacteria, and extremophiles), yeast and/or mixtures of these. In someinstances, microbial biomass can be obtained from natural sources, e.g.,the ocean, lakes, bodies of water, e.g., salt water or fresh water, oron land. Alternatively or in addition, microbial biomass can be obtainedfrom culture systems, e.g., large scale dry and wet culture systems.

Blends of any biomass materials described herein can be utilized formaking any of the intermediates or products described herein. Forexample, blends of cellulosic materials and starchy materials can beutilized for making any product described herein.

Systems for Treating Biomass and Using Treated Biomass in Fermentation

FIG. 1 shows a system 100 for treating biomass, particularly fibrousbiomass, and then using the treated biomass to enhance a fermentationprocess. System 100 includes a module 102 in which a biomass feedstockis mechanically treated, e.g., exposing internal fibers of thefeedstock. Examples of mechanical treatments will be described in detailbelow. The system 100 also includes a module 104 in which themechanically treated feedstock is functionalized, e.g., by irradiation.After functionalization, the functionalized fibers are delivered to afermentation system 106 by a delivery module 108.

The functionalized fibers are then present during fermentation andenhance the fermentation process by providing a substrate that caninteract with the microorganisms used in fermentation, e.g., yeastcells. This interaction is shown schematically in FIG. 2, which depictsa functionalized polar fiber 10 and a yeast cell 12 having acomplementary polar functional group. Due to the polarity of the fibersand the yeast cell, the cell can become immobilized on one or more ofthe fibers. Bonding of the yeast cell (or other microorganism) to thefibers may be by hydrogen bonding, or by covalent or ionic bonding. Insome cases, the functional groups on the fibers may react with those onthe microorganism, forming a covalent bond. The increased surface areaand porosity of the biomass material that results from mechanicaltreatment (e.g., in module 102) provides a greater surface area forinteraction of the fiber and microorganism and thus enhances thisinteraction. The immobilized cells are more productive, increasing theefficiency and yield of the fermentation process and preventing theprocess from becoming prematurely “stuck.”

It is noted that if mixing is performed during fermentation, the mixingis preferably relatively gentle (low shear) so as to minimize disruptionof the interaction between the microorganisms and fibers. In someembodiments, jet mixing is used, as described in U.S. Ser. No.61/218,832 and U.S. Ser. No. 61/179,995, the complete disclosures ofwhich are incorporated herein by reference.

Referring again to FIG. 1, fermentation produces a crude ethanolmixture, which flows into a holding tank 110. Water or other solvent,and other non-ethanol components, are stripped from the crude ethanolmixture using a stripping column 112, and the ethanol is then distilledusing a distillation unit 114, e.g., a rectifier. Finally, the ethanolcan be dried using a molecular sieve 116, denatured if necessary, andoutput to a desired shipping method.

In some cases, the systems described herein, or components thereof, maybe portable, so that the system can be transported (e.g., by rail,truck, or marine vessel) from one location to another. The method stepsdescribed herein can be performed at one or more locations, and in somecases one or more of the steps can be performed in transit. Such mobileprocessing is described in U.S. Ser. No. 12/374,549 and InternationalApplication No. WO 2008/011598, the full disclosures of which areincorporated herein by reference.

Any or all of the method steps described herein can be performed atambient temperature. If desired, cooling and/or heating may be employedduring certain steps. For example, the feedstock may be cooled duringmechanical treatment to increase its brittleness. In some embodiments,cooling is employed before, during or after the initial mechanicaltreatment and/or the subsequent mechanical treatment. Cooling may beperformed as described in Ser. No. 12/502,629, the full disclosure ofwhich is incorporated herein by reference. Moreover, the temperature inthe fermentation system 106 may be controlled to enhance fermentation.

Physical Treatment

Physical treatment processes that may be used to change the morphologyof the biomass material and/or to functionalize the material can includeone or more of any of those described herein, such as mechanicaltreatment, chemical treatment, irradiation, sonication, oxidation,pyrolysis or steam explosion. Treatment methods can be used incombinations of two, three, four, or even all of these technologies (inany order). When more than one treatment method is used, the methods canbe applied at the same time or at different times. Other processes thatfunctionalize a biomass feedstock and/or alter its morphology may alsobe used, alone or in combination with the processes disclosed herein.

Mechanical Treatments

In some cases, methods can include mechanically treating the biomassfeedstock. Mechanical treatments include, for example, cutting, milling,pressing, grinding, shearing and chopping. Milling may include, forexample, ball milling, hammer milling, rotor/stator dry or wet milling,or other types of milling. Other mechanical treatments include, e.g.,stone grinding, cracking, mechanical ripping or tearing, pin grinding orair attrition milling.

Mechanical treatment can be advantageous for “opening up,” “stressing,”breaking and shattering the cellulosic or lignocellulosic materials,making the cellulose of the materials more susceptible to chain scissionand/or reduction of crystallinity. The open materials can also be moresusceptible to oxidation when irradiated.

In some cases, the mechanical treatment may include an initialpreparation of the feedstock as received, e.g., size reduction ofmaterials, such as by cutting, grinding, shearing, pulverizing orchopping. For example, in some cases, loose feedstock (e.g., recycledpaper, starchy materials, or switchgrass) is prepared by shearing orshredding.

Alternatively, or in addition, the feedstock material can first bephysically treated by one or more of the other physical treatmentmethods, e.g., chemical treatment, radiation, sonication, oxidation,pyrolysis or steam explosion, and then mechanically treated. Thissequence can be advantageous since materials treated by one or more ofthe other treatments, e.g., irradiation or pyrolysis, tend to be morebrittle and, therefore, it may be easier to further change the molecularstructure of the material by mechanical treatment.

In some embodiments, the biomass material is fibrous, and mechanicaltreatment includes shearing to expose fibers of the fibrous material.Shearing can be performed, for example, using a rotary knife cutter.Other methods of mechanically treating the biomass include, for example,milling or grinding. Milling may be performed using, for example, ahammer mill, ball mill, colloid mill, conical or cone mill, disk mill,edge mill, Wiley mill or grist mill. Grinding may be performed using,for example, a stone grinder, pin grinder, coffee grinder, or bungrinder. Grinding may be provided, for example, by a reciprocating pinor other element, as is the case in a pin mill. Other mechanicaltreatment methods include mechanical ripping or tearing, other methodsthat apply pressure to the material, and air attrition milling. Suitablemechanical treatments further include any other technique that changesthe molecular structure or morphology of the biomass material.

If desired, the mechanically treated material can be passed through ascreen, e.g., having an average opening size of 1.59 mm or less ( 1/16inch, 0.0625 inch). In some embodiments, shearing, or other mechanicaltreatment, and screening are performed concurrently. For example, arotary knife cutter can be used to concurrently shear and screen thebiomass material. The biomass is sheared between stationary blades androtating blades to provide a sheared material that passes through ascreen, and is captured in a bin.

The biomass material can be mechanically treated in a dry state (e.g.,having little or no free water on its surface), a hydrated state (e.g.,having up to ten percent by weight absorbed water), or in a wet state,e.g., having between about 10 percent and about 75 percent by weightwater. The biomass material can even be mechanically treated whilepartially or fully submerged under a liquid, such as water, ethanol orisopropanol. The biomass material can also be mechanically treated undera gas (such as a stream or atmosphere of gas other than air), e.g.,oxygen or nitrogen, or steam.

Mechanical treatment systems can be configured to produce streams withspecific morphology characteristics such as, for example, surface area,porosity, bulk density, and, in the case of fibrous feedstocks, fibercharacteristics such as length-to-width ratio.

In some embodiments, a BET surface area of the mechanically treatedmaterial is greater than 0.1 m²/g, e.g., greater than 0.25 m²/g, greaterthan 0.5 m²/g, greater than 1.0 m²/g, greater than 1.5 m²/g, greaterthan 1.75 m²/g, greater than 5.0 m²/g, greater than 10 m²/g, greaterthan 25 m²/g, greater than 35 m²/g, greater than 50 m²/g, greater than60 m²/g, greater than 75 m²/g, greater than 100 m²/g, greater than 150m²/g, greater than 200 m²/g, or even greater than 250 m²/g.

A porosity of the mechanically treated material can be, e.g., greaterthan 20 percent, greater than 25 percent, greater than 35 percent,greater than 50 percent, greater than 60 percent, greater than 70percent, greater than 80 percent, greater than 85 percent, greater than90 percent, greater than 92 percent, greater than 94 percent, greaterthan 95 percent, greater than 97.5 percent, greater than 99 percent, oreven greater than 99.5 percent.

In some embodiments, after mechanical treatment the material has a bulkdensity of less than 0.25 g/cm³, e.g., 0.20 g/cm³, 0.15 g/cm³, 0.10g/cm³, 0.05 g/cm³ or less, e.g., 0.025 g/cm³. Bulk density is determinedusing ASTM D1895B. Briefly, the method involves filling a measuringcylinder of known volume with a sample and obtaining a weight of thesample. The bulk density is calculated by dividing the weight of thesample in grams by the known volume of the cylinder in cubiccentimeters.

If the biomass is a fibrous material the fibers of the mechanicallytreated material can have a relatively large average length-to-diameterratio (e.g., greater than 20-to-1), even if they have been sheared morethan once. In addition, the fibers of the fibrous materials describedherein may have a relatively narrow length and/or length-to-diameterratio distribution.

As used herein, average fiber widths (e.g., diameters) are thosedetermined optically by randomly selecting approximately 5,000 fibers.Average fiber lengths are corrected length-weighted lengths. BET(Brunauer, Emmet and Teller) surface areas are multi-point surfaceareas, and porosities are those determined by mercury porosimetry.

If the biomass is a fibrous material the average length-to-diameterratio of fibers of the mechanically treated material can be, e.g.,greater than 8/1, e.g., greater than 10/1, greater than 15/1, greaterthan 20/1, greater than 25/1, or greater than 50/1. An average fiberlength of the mechanically treated material can be, e.g., between about0.5 mm and 2.5 mm, e.g., between about 0.75 mm and 1.0 mm, and anaverage width (e.g., diameter) of the second fibrous material 14 can be,e.g., between about 5 μm and 50 μm, e.g., between about 10 μm and 30 μm.

In some embodiments, if the biomass is a fibrous material the standarddeviation of the fiber length of the mechanically treated material canbe less than 60 percent of an average fiber length of the mechanicallytreated material, e.g., less than 50 percent of the average length, lessthan 40 percent of the average length, less than 25 percent of theaverage length, less than 10 percent of the average length, less than 5percent of the average length, or even less than 1 percent of theaverage length.

In some situations, it can be desirable to prepare a low bulk densitymaterial, densify the material (e.g., to make it easier and less costlyto transport to another site), and then revert the material to a lowerbulk density state. Densified materials can be processed by any of themethods described herein, or any material processed by any of themethods described herein can be subsequently densified, e.g., asdisclosed in U.S. Ser. No. 12/429,045 and WO 2008/073186, the fulldisclosures of which are incorporated herein by reference.

Radiation Treatment

One or more radiation processing sequences can be used to process thebiomass, e.g., to functionalize the material. Radiation can alsosterilize the materials, or any media needed to bioprocess the material.

In some embodiments, energy deposited in a material that releases anelectron from its atomic orbital is used to irradiate the materials. Theradiation may be provided by (1) heavy charged particles, such as alphaparticles or protons, (2) electrons, produced, for example, in betadecay or electron beam accelerators, or (3) electromagnetic radiation,for example, gamma rays, x rays, or ultraviolet rays. In one approach,radiation produced by radioactive substances can be used to irradiatethe feedstock. In another approach, electromagnetic radiation (e.g.,produced using electron beam emitters) can be used to irradiate thefeedstock. In some embodiments, any combination in any order orconcurrently of (1) through (3) may be utilized. The doses applieddepend on the desired effect and the particular feedstock.

In some instances when chain scission is desirable and/or polymer chainfunctionalization is desirable, particles heavier than electrons, suchas protons, helium nuclei, argon ions, silicon ions, neon ions, carbonions, phoshorus ions, oxygen ions or nitrogen ions can be utilized. Whenring-opening chain scission is desired, positively charged particles canbe utilized for their Lewis acid properties for enhanced ring-openingchain scission. For example, when maximum oxidation is desired, oxygenions can be utilized, and when maximum nitration is desired, nitrogenions can be utilized. The use of heavy particles and positively chargedparticles is described in U.S. Ser. No. 12/417,699, the full disclosureof which is incorporated herein by reference.

In one method, a first material that is or includes cellulose having afirst number average molecular weight (M_(N1)) is irradiated, e.g., bytreatment with ionizing radiation (e.g., in the form of gamma radiation,X-ray radiation, 100 nm to 280 nm ultraviolet (UV) light, a beam ofelectrons or other charged particles) to provide a second material thatincludes cellulose having a second number average molecular weight(M_(N2)) lower than the first number average molecular weight. Thesecond material (or the first and second material) can be combined witha microorganism (with or without enzyme treatment) that can utilize thesecond and/or first material or its constituent sugars or lignin toproduce an intermediate or product, such as those described herein.

Since the second material includes cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable and/or soluble, e.g., in a solution containing amicroorganism and/or an enzyme. These properties make the secondmaterial easier to process and more susceptible to chemical, enzymaticand/or biological attack relative to the first material, which cangreatly improve the production rate and/or production level of a desiredproduct, e.g., ethanol.

In some embodiments, the second number average molecular weight (M_(N2))is lower than the first number average molecular weight (M_(N1)) by morethan about 10 percent, e.g., more than about 15, 20, 25, 30, 35, 40, 50percent, 60 percent, or even more than about 75 percent.

In some instances, the second material includes cellulose that has acrystallinity (C₂) that is lower than the crystallinity (C₁) of thecellulose of the first material. For example, (C₂) can be lower than(C₁) by more than about 10 percent, e.g., more than about 15, 20, 25,30, 35, 40, or even more than about 50 percent.

In some embodiments, the starting crystallinity index (prior toirradiation) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after irradiation is from about 10 to about 50percent, e.g., from about 15 to about 45 percent or from about 20 toabout 40 percent. However, in some embodiments, e.g., after extensiveirradiation, it is possible to have a crystallinity index of lower than5 percent. In some embodiments, the material after irradiation issubstantially amorphous.

In some embodiments, the starting number average molecular weight (priorto irradiation) is from about 200,000 to about 3,200,000, e.g., fromabout 250,000 to about 1,000,000 or from about 250,000 to about 700,000,and the number average molecular weight after irradiation is from about50,000 to about 200,000, e.g., from about 60,000 to about 150,000 orfrom about 70,000 to about 125,000. However, in some embodiments, e.g.,after extensive irradiation, it is possible to have a number averagemolecular weight of less than about 10,000 or even less than about5,000.

In some embodiments, the second material can have a level of oxidation(O₂) that is higher than the level of oxidation (O₁) of the firstmaterial. A higher level of oxidation of the material can aid in itsdispersability, swellability and/or solubility, further enhancing thematerial's susceptibility to chemical, enzymatic or biological attack.In some embodiments, to increase the level of the oxidation of thesecond material relative to the first material, the irradiation isperformed under an oxidizing environment, e.g., under a blanket of airor oxygen, producing a second material that is more oxidized than thefirst material. For example, the second material can have more hydroxylgroups, aldehyde groups, ketone groups, ester groups or carboxylic acidgroups, which can increase its hydrophilicity.

Ionizing Radiation

Each form of radiation ionizes the carbon-containing material viaparticular interactions, as determined by the energy of the radiation.Heavy charged particles primarily ionize matter via Coulomb scattering;furthermore, these interactions produce energetic electrons that mayfurther ionize matter. Alpha particles are identical to the nucleus of ahelium atom and are produced by the alpha decay of various radioactivenuclei, such as isotopes of bismuth, polonium, astatine, radon,francium, radium, several actinides, such as actinium, thorium, uranium,neptunium, curium, californium, americium, and plutonium.

When particles are utilized, they can be neutral (uncharged), positivelycharged or negatively charged. When charged, the charged particles canbear a single positive or negative charge, or multiple charges, e.g.,one, two, three or even four or more charges. In instances in whichchain scission is desired, positively charged particles may bedesirable, in part due to their acidic nature. When particles areutilized, the particles can have the mass of a resting electron, orgreater, e.g., 500, 1000, 1500, 2000, 10,000 or even 100,000 times themass of a resting electron. For example, the particles can have a massof from about 1 atomic unit to about 150 atomic units, e.g., from about1 atomic unit to about 50 atomic units, or from about 1 to about 25,e.g., 1, 2, 3, 4, 5, 10, 12 or 15 amu. Accelerators used to acceleratethe particles can be electrostatic DC, electrodynamic DC, RF linear,magnetic induction linear or continuous wave. For example, cyclotrontype accelerators are available from IBA, Belgium, such as theRhodotron® system, while DC type accelerators are available from RDI,now IBA Industrial, such as the Dynamitron®. Ions and ion acceleratorsare discussed in Introductory Nuclear Physics, Kenneth S. Krane, JohnWiley & Sons, Inc. (1988), Krsto Prelec, FIZIKA B 6 (1997) 4, 177-206,Chu, William T., “Overview of Light-Ion Beam Therapy” Columbus-Ohio,ICRU-IAEA Meeting, 18-20 Mar. 2006, Iwata, Y. et al.,“Alternating-Phase-Focused IH-DTL for Heavy-Ion Medical Accelerators”Proceedings of EPAC 2006, Edinburgh, Scotland and Leaner, C. M. et al.,“Status of the Superconducting ECR Ion Source Venus” Proceedings of EPAC2000, Vienna, Austria.

Gamma radiation has the advantage of a significant penetration depthinto a variety of materials. Sources of gamma rays include radioactivenuclei, such as isotopes of cobalt, calcium, technicium, chromium,gallium, indium, iodine, iron, krypton, samarium, selenium, sodium,thalium, and xenon.

Sources of x rays include electron beam collision with metal targets,such as tungsten or molybdenum or alloys, or compact light sources, suchas those produced commercially by Lyncean.

Sources for ultraviolet radiation include deuterium or cadmium lamps.

Sources for infrared radiation include sapphire, zinc, or selenidewindow ceramic lamps.

Sources for microwaves include klystrons, Slevin type RF sources, oratom beam sources that employ hydrogen, oxygen, or nitrogen gases.

In some embodiments, a beam of electrons is used as the radiationsource. A beam of electrons has the advantages of high dose rates (e.g.,1, 5, or even 10 Mrad per second), high throughput, less containment,and less confinement equipment. Electrons can also be more efficient atcausing chain scission. In addition, electrons having energies of 4-10MeV can have a penetration depth of 5 to 30 mm or more, such as 40 mm.

Electron beams can be generated, e.g., by electrostatic generators,cascade generators, transformer generators, low energy accelerators witha scanning system, low energy accelerators with a linear cathode, linearaccelerators, and pulsed accelerators. Electrons as an ionizingradiation source can be useful, e.g., for relatively thin sections ofmaterial, e.g., less than 0.5 inch, e.g., less than 0.4 inch, 0.3 inch,0.2 inch, or less than 0.1 inch. In some embodiments, the energy of eachelectron of the electron beam is from about 0.3 MeV to about 2.0 MeV(million electron volts), e.g., from about 0.5 MeV to about 1.5 MeV, orfrom about 0.7 MeV to about 1.25 MeV.

Electron beam irradiation devices may be procured commercially from IonBeam Applications, Louvain-la-Neuve, Belgium or the Titan Corporation,San Diego, Calif. Typical electron energies can be 1 MeV, 2 MeV, 4.5MeV, 7.5 MeV, or 10 MeV. Typical electron beam irradiation device powercan be 1 kW, 5 kW, 10 kW, 20 kW, 50 kW, 100 kW, 250 kW, or 500 kW. Thelevel of depolymerization of the feedstock depends on the electronenergy used and the dose applied, while exposure time depends on thepower and dose. Typical doses may take values of 1 kGy, 5 kGy, 10 kGy,20 kGy, 50 kGy, 100 kGy, or 200 kGy.

Ion Particle Beams

Particles heavier than electrons can be utilized to irradiate any of thebiomass materials described herein. For example, protons, helium nuclei,argon ions, silicon ions, neon ions carbon ions, phoshorus ions, oxygenions or nitrogen ions can be utilized. In some embodiments, particlesheavier than electrons can induce higher amounts of chain scission(relative to lighter particles). In some instances, positively chargedparticles can induce higher amounts of chain scission than negativelycharged particles due to their acidity.

Heavier particle beams can be generated, e.g., using linear acceleratorsor cyclotrons. In some embodiments, the energy of each particle of thebeam is from about 1.0 MeV/atomic unit to about 6,000 MeV/atomic unit,e.g., from about 3 MeV/atomic unit to about 4,800 MeV/atomic unit, orfrom about 10 MeV/atomic unit to about 1,000 MeV/atomic unit.

In certain embodiments, ion beams used to irradiate carbon-containingmaterials, e.g., biomass materials, can include more than one type ofion. For example, ion beams can include mixtures of two or more (e.g.,three, four or more) different types of ions. Exemplary mixtures caninclude carbon ions and protons, carbon ions and oxygen ions, nitrogenions and protons, and iron ions and protons. More generally, mixtures ofany of the ions discussed above (or any other ions) can be used to formirradiating ion beams. In particular, mixtures of relatively light andrelatively heavier ions can be used in a single ion beam.

In some embodiments, ion beams for irradiating materials includepositively-charged ions. The positively charged ions can include, forexample, positively charged hydrogen ions (e.g., protons), noble gasions (e.g., helium, neon, argon), carbon ions, nitrogen ions, oxygenions, silicon atoms, phosphorus ions, and metal ions such as sodiumions, calcium ions, and/or iron ions. Without wishing to be bound by anytheory, it is believed that such positively-charged ions behavechemically as Lewis acid moieties when exposed to materials, initiatingand sustaining cationic ring-opening chain scission reactions in anoxidative environment.

In certain embodiments, ion beams for irradiating materials includenegatively-charged ions. Negatively charged ions can include, forexample, negatively charged hydrogen ions (e.g., hydride ions), andnegatively charged ions of various relatively electronegative nuclei(e.g., oxygen ions, nitrogen ions, carbon ions, silicon ions, andphosphorus ions). Without wishing to be bound by any theory, it isbelieved that such negatively-charged ions behave chemically as Lewisbase moieties when exposed to materials, causing anionic ring-openingchain scission reactions in a reducing environment.

In some embodiments, beams for irradiating materials can include neutralatoms. For example, any one or more of hydrogen atoms, helium atoms,carbon atoms, nitrogen atoms, oxygen atoms, neon atoms, silicon atoms,phosphorus atoms, argon atoms, and iron atoms can be included in beamsthat are used for irradiation of biomass materials. In general, mixturesof any two or more of the above types of atoms (e.g., three or more,four or more, or even more) can be present in the beams.

In certain embodiments, ion beams used to irradiate materials includesingly-charged ions such as one or more of H⁺, H⁻, He⁺, Ne⁺, Ar⁺, C⁺,C⁻, O⁺, O⁻, N⁺, N⁻, Si⁺, Si⁻, P⁺, P⁻, Na⁺, Ca⁺, and Fe⁺. In someembodiments, ion beams can include multiply-charged ions such as one ormore of C²⁺, C³⁺, C⁴⁺, N³⁺, N⁵⁺, N³⁻, O²⁺, O²⁻, O₂ ²⁻, Si²⁺, Si⁴⁺, Si²⁻,and Si⁴⁻. In general, the ion beams can also include more complexpolynuclear ions that bear multiple positive or negative charges. Incertain embodiments, by virtue of the structure of the polynuclear ion,the positive or negative charges can be effectively distributed oversubstantially the entire structure of the ions. In some embodiments, thepositive or negative charges can be somewhat localized over portions ofthe structure of the ions.

Electromagnetic Radiation

In embodiments in which the irradiating is performed withelectromagnetic radiation, the electromagnetic radiation can have, e.g.,energy per photon (in electron volts) of greater than 10² eV, e.g.,greater than 10³, 10⁴, 10⁵, 10⁶, or even greater than 10⁷ eV. In someembodiments, the electromagnetic radiation has energy per photon ofbetween 10⁴ and 10⁷, e.g., between 10⁵ and 10⁶ eV. The electromagneticradiation can have a frequency of, e.g., greater than 10¹⁶ hz, greaterthan 10¹⁷ hz, 10¹⁸, 10¹⁹, 10²⁰, or even greater than 10²¹ hz. In someembodiments, the electromagnetic radiation has a frequency of between10¹⁸ and 10²² hz, e.g., between 10¹⁹ to 10²¹ hz.

Quenching and Controlled Functionalization of Biomass

After treatment with ionizing radiation, any of the materials ormixtures described herein may become ionized; that is, the treatedmaterial may include radicals at levels that are detectable with anelectron spin resonance spectrometer. If ionized biomass remains in theatmosphere, it will be oxidized, such as to an extent that carboxylicacid groups are generated by reacting with the atmospheric oxygen. Insome instances with some materials, such oxidation is desired because itcan aid in the further breakdown in molecular weight of thecarbohydrate-containing biomass, and the oxidation groups, e.g.,carboxylic acid groups can be helpful for solubility and microorganismutilization in some instances. However, since the radicals can “live”for some time after irradiation, e.g., longer than 1 day, 5 days, 30days, 3 months, 6 months or even longer than 1 year, material propertiescan continue to change over time, which in some instances, can beundesirable. Thus, it may be desirable to quench the ionized material.

After ionization, any biomass material that has been ionized can bequenched to reduce the level of radicals in the ionized biomass, e.g.,such that the radicals are no longer detectable with the electron spinresonance spectrometer. For example, the radicals can be quenched by theapplication of a sufficient pressure to the biomass and/or by utilizinga fluid in contact with the ionized biomass, such as a gas or liquid,that reacts with (quenches) the radicals. Using a gas or liquid to atleast aid in the quenching of the radicals can be used to functionalizethe ionized biomass with a desired amount and kind of functional groups,such as carboxylic acid groups, enol groups, aldehyde groups, nitrogroups, nitrile groups, amino groups, alkyl amino groups, alkyl groups,chloroalkyl groups or chlorofluoroalkyl groups.

In some instances, such quenching can improve the stability of some ofthe ionized biomass materials. For example, quenching can improve theresistance of the biomass to oxidation. Functionalization by quenchingcan also improve the solubility of any biomass described herein, canimprove its thermal stability, and can improve material utilization byvarious microorganisms. For example, the functional groups imparted tothe biomass material by the quenching can act as receptor sites forattachment by microorganisms, e.g., to enhance cellulose hydrolysis byvarious microorganisms.

In some embodiments, quenching includes an application of pressure tothe biomass, such as by mechanically deforming the biomass, e.g.,directly mechanically compressing the biomass in one, two, or threedimensions, or applying pressure to a fluid in which the biomass isimmersed, e.g., isostatic pressing. In such instances, the deformationof the material itself brings radicals, which are often trapped incrystalline domains, in close enough proximity so that the radicals canrecombine, or react with another group. In some instances, the pressureis applied together with the application of heat, such as a sufficientquantity of heat to elevate the temperature of the biomass to above amelting point or softening point of a component of the biomass, such aslignin, cellulose or hemicellulose. Heat can improve molecular mobilityin the material, which can aid in the quenching of the radicals. Whenpressure is utilized to quench, the pressure can be greater than about1000 psi, such as greater than about 1250 psi, 1450 psi, 3625 psi, 5075psi, 7250 psi, 10000 psi or even greater than 15000 psi.

In some embodiments, quenching includes contacting the biomass with afluid, such as a liquid or gas, e.g., a gas capable of reacting with theradicals, such as acetylene or a mixture of acetylene in nitrogen,ethylene, chlorinated ethylenes or chlorofluoroethylenes, propylene ormixtures of these gases. In other particular embodiments, quenchingincludes contacting the biomass with a liquid, e.g., a liquid solublein, or at least capable of penetrating into the biomass and reactingwith the radicals, such as a diene, such as 1,5-cyclooctadiene. In somespecific embodiments, quenching includes contacting the biomass with anantioxidant, such as Vitamin E. If desired, the biomass feedstock caninclude an antioxidant dispersed therein, and the quenching can comefrom contacting the antioxidant dispersed in the biomass feedstock withthe radicals.

Functionalization can be enhanced by utilizing heavy charged ions, suchas any of the heavier ions described herein. For example, if it isdesired to enhance oxidation, charged oxygen ions can be utilized forthe irradiation. If nitrogen functional groups are desired, nitrogenions or anions that include nitrogen can be utilized. Likewise, ifsulfur or phosphorus groups are desired, sulfur or phosphorus ions canbe used in the irradiation.

Doses

In some instances, the irradiation is performed at a dosage rate ofgreater than about 0.25 Mrad per second, e.g., greater than about 0.5,0.75, 1.0, 1.5, 2.0, or even greater than about 2.5 Mrad per second. Insome embodiments, the irradiating is performed at a dose rate of between5.0 and 1500.0 kilorads/hour, e.g., between 10.0 and 750.0 kilorads/houror between 50.0 and 350.0 kilorads/hour.

In some embodiments, the irradiating (with any radiation source or acombination of sources) is performed until the material receives a doseof at least 0.1 Mrad, at least 0.25 Mrad, e.g., at least 1.0 Mrad, atleast 2.5 Mrad, at least 5.0 Mrad, at least 10.0 Mrad, at least 60 Mrador at least 100 Mrad. In some embodiments, the irradiating is performeduntil the material receives a dose of from about 0.1 Mrad to about 500Mrad, from about 0.5 Mrad to about 200 Mrad, from about 1 Mrad to about100 Mrad, or from about 5 Mrad to about 60 Mrad. In some embodiments, arelatively low dose of radiation is applied, e.g., less than 60 Mrad.

Sonication

Sonication can reduce the molecular weight and/or crystallinity ofmaterials, such as one or more of any of the materials described herein,e.g., one or more carbohydrate sources, such as cellulosic orlignocellulosic materials, or starchy materials. Sonication can also beused to sterilize the materials.

In one method, a first material that includes cellulose having a firstnumber average molecular weight (M_(N1)) is dispersed in a medium, suchas water, and sonicated and/or otherwise cavitated, to provide a secondmaterial that includes cellulose having a second number averagemolecular weight (M_(N2)) lower than the first number average molecularweight. The second material (or the first and second material in certainembodiments) can be combined with a microorganism (with or withoutenzyme treatment) that can utilize the second and/or first material toproduce an intermediate or product.

Since the second material includes cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable, and/or soluble, e.g., in a solution containing amicroorganism.

In some embodiments, the second number average molecular weight (M_(N2))is lower than the first number average molecular weight (M_(N1)) by morethan about 10 percent, e.g., more than about 15, 20, 25, 30, 35, 40, 50percent, 60 percent, or even more than about 75 percent.

In some instances, the second material includes cellulose that has acrystallinity (C₂) that is lower than the crystallinity (C_(N1)) of thecellulose of the first material. For example, (C₂) can be lower than(C_(N1)) by more than about 10 percent, e.g., more than about 15, 20,25, 30, 35, 40, or even more than about 50 percent.

In some embodiments, the starting crystallinity index (prior tosonication) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after sonication is from about 10 to about 50percent, e.g., from about 15 to about 45 percent or from about 20 toabout 40 percent. However, in certain embodiments, e.g., after extensivesonication, it is possible to have a crystallinity index of lower than 5percent. In some embodiments, the material after sonication issubstantially amorphous.

In some embodiments, the starting number average molecular weight (priorto sonication) is from about 200,000 to about 3,200,000, e.g., fromabout 250,000 to about 1,000,000 or from about 250,000 to about 700,000,and the number average molecular weight after sonication is from about50,000 to about 200,000, e.g., from about 60,000 to about 150,000 orfrom about 70,000 to about 125,000. However, in some embodiments, e.g.,after extensive sonication, it is possible to have a number averagemolecular weight of less than about 10,000 or even less than about5,000.

In some embodiments, the second material can have a level of oxidation(O₂) that is higher than the level of oxidation (O₁) of the firstmaterial. A higher level of oxidation of the material can aid in itsdispersability, swellability and/or solubility, further enhancing thematerial's susceptibility to chemical, enzymatic or microbial attack. Insome embodiments, to increase the level of the oxidation of the secondmaterial relative to the first material, the sonication is performed inan oxidizing medium, producing a second material that is more oxidizedthan the first material. For example, the second material can have morehydroxyl groups, aldehyde groups, ketone groups, ester groups orcarboxylic acid groups, which can increase its hydrophilicity.

In some embodiments, the sonication medium is an aqueous medium. Ifdesired, the medium can include an oxidant, such as a peroxide (e.g.,hydrogen peroxide), a dispersing agent and/or a buffer. Examples ofdispersing agents include ionic dispersing agents, e.g., sodium laurylsulfate, and non-ionic dispersing agents, e.g., poly(ethylene glycol).

In other embodiments, the sonication medium is non-aqueous. For example,the sonication can be performed in a hydrocarbon, e.g., toluene orheptane, an ether, e.g., diethyl ether or tetrahydrofuran, or even in aliquefied gas such as argon, xenon, or nitrogen.

Pyrolysis

One or more pyrolysis processing sequences can be used to physicallytreat the biomass material. Pyrolysis can also be used to sterilize thematerial.

In one example, a first material that includes cellulose having a firstnumber average molecular weight (M_(N1)) is pyrolyzed, e.g., by heatingthe first material in a tube furnace (in the presence or absence ofoxygen), to provide a second material that includes cellulose having asecond number average molecular weight (M_(N2)) lower than the firstnumber average molecular weight.

Since the second material includes cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable and/or soluble, e.g., in a solution containing amicroorganism.

In some embodiments, the second number average molecular weight (M_(N2))is lower than the first number average molecular weight (M_(N1)) by morethan about 10 percent, e.g., more than about 15, 20, 25, 30, 35, 40, 50percent, 60 percent, or even more than about 75 percent.

In some instances, the second material includes cellulose that has acrystallinity (C₂) that is lower than the crystallinity (C_(N1)) of thecellulose of the first material. For example, (C₂) can be lower than(C_(N1)) by more than about 10 percent, e.g., more than about 15, 20,25, 30, 35, 40, or even more than about 50 percent.

In some embodiments, the starting crystallinity (prior to pyrolysis) isfrom about 40 to about 87.5 percent, e.g., from about 50 to about 75percent or from about 60 to about 70 percent, and the crystallinityindex after pyrolysis is from about 10 to about 50 percent, e.g., fromabout 15 to about 45 percent or from about 20 to about 40 percent.However, in certain embodiments, e.g., after extensive pyrolysis, it ispossible to have a crystallinity index of lower than 5 percent. In someembodiments, the material after pyrolysis is substantially amorphous.

In some embodiments, the starting number average molecular weight (priorto pyrolysis) is from about 200,000 to about 3,200,000, e.g., from about250,000 to about 1,000,000 or from about 250,000 to about 700,000, andthe number average molecular weight after pyrolysis is from about 50,000to about 200,000, e.g., from about 60,000 to about 150,000 or from about70,000 to about 125,000. However, in some embodiments, e.g., afterextensive pyrolysis, it is possible to have a number average molecularweight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second material can have a level of oxidation(O₂) that is higher than the level of oxidation (O₁) of the firstmaterial. A higher level of oxidation of the material can aid in itsdispersability, swellability and/or solubility, further enhancing thesusceptibility of the material to chemical, enzymatic or microbialattack. In some embodiments, to increase the level of the oxidation ofthe second material relative to the first material, the pyrolysis isperformed in an oxidizing environment, producing a second material thatis more oxidized than the first material. For example, the secondmaterial can have more hydroxyl groups, aldehyde groups, ketone groups,ester groups or carboxylic acid groups, than the first material, therebyincreasing the hydrophilicity of the material.

In some embodiments, the pyrolysis of the materials is continuous. Inother embodiments, the material is pyrolyzed for a pre-determined time,and then allowed to cool for a second pre-determined time beforepyrolyzing again.

Oxidation

One or more oxidative processing sequences can be used to physicallytreat the biomass material. The oxidation conditions can be varied,e.g., depending on the lignin content of the feedstock, with a higherdegree of oxidation generally being desired for higher lignin contentfeedstocks.

In one method, a first material that includes cellulose having a firstnumber average molecular weight (M_(N1)) and having a first oxygencontent (O₁) is oxidized, e.g., by heating the first material in astream of air or oxygen-enriched air, to provide a second material thatincludes cellulose having a second number average molecular weight(M_(N2)) and having a second oxygen content (O₂) higher than the firstoxygen content (O₁).

The second number average molecular weight of the second material isgenerally lower than the first number average molecular weight of thefirst material. For example, the molecular weight may be reduced to thesame extent as discussed above with respect to the other physicaltreatments. The crystallinity of the second material may also be reducedto the same extent as discussed above with respect to the other physicaltreatments.

In some embodiments, the second oxygen content is at least about fivepercent higher than the first oxygen content, e.g., 7.5 percent higher,10.0 percent higher, 12.5 percent higher, 15.0 percent higher or 17.5percent higher. In some preferred embodiments, the second oxygen contentis at least about 20.0 percent higher than the first oxygen content ofthe first material. Oxygen content is measured by elemental analysis bypyrolyzing a sample in a furnace operating at 1300° C. or higher. Asuitable elemental analyzer is the LECO CHNS-932 analyzer with a VTF-900high temperature pyrolysis furnace.

Generally, oxidation of a material occurs in an oxidizing environment.For example, the oxidation can be effected or aided by pyrolysis in anoxidizing environment, such as in air or argon enriched in air. To aidin the oxidation, various chemical agents, such as oxidants, acids orbases can be added to the material prior to or during oxidation. Forexample, a peroxide (e.g., benzoyl peroxide) can be added prior tooxidation.

Some oxidative methods of reducing recalcitrance in a biomass feedstockemploy Fenton-type chemistry. Such methods are disclosed, for example,in U.S. Ser. No. 12/639,289, the complete disclosure of which isincorporated herein by reference.

Exemplary oxidants include peroxides, such as hydrogen peroxide andbenzoyl peroxide, persulfates, such as ammonium persulfate, activatedforms of oxygen, such as ozone, permanganates, such as potassiumpermanganate, perchlorates, such as sodium perchlorate, andhypochlorites, such as sodium hypochlorite (household bleach).

In some situations, pH is maintained at or below about 5.5 duringcontact, such as between 1 and 5, between 2 and 5, between 2.5 and 5 orbetween about 3 and 5. Oxidation conditions can also include a contactperiod of between 2 and 12 hours, e.g., between 4 and 10 hours orbetween 5 and 8 hours. In some instances, temperature is maintained ator below 300° C., e.g., at or below 250, 200, 150, 100 or 50° C. In someinstances, the temperature remains substantially ambient, e.g., at orabout 20-25° C.

In some embodiments, the one or more oxidants are applied as a gas, suchas by generating ozone in-situ by irradiating the material through airwith a beam of particles, such as electrons.

In some embodiments, the mixture further includes one or morehydroquinones, such as 2,5-dimethoxyhydroquinone (DMHQ) and/or one ormore benzoquinones, such as 2,5-dimethoxy-1,4-benzoquinone (DMBQ), whichcan aid in electron transfer reactions.

In some embodiments, the one or more oxidants areelectrochemically-generated in-situ. For example, hydrogen peroxideand/or ozone can be electro-chemically produced within a contact orreaction vessel.

Other Processes to Functionalize

Any of the processes of this paragraph can be used alone without any ofthe processes described herein, or in combination with any of theprocesses described herein (in any order): steam explosion, chemicaltreatment (e.g., acid treatment (including concentrated and dilute acidtreatment with mineral acids, such as sulfuric acid, hydrochloric acidand organic acids, such as trifluoroacetic acid) and/or base treatment(e.g., treatment with lime or sodium hydroxide)), UV treatment, screwextrusion treatment (see, e.g., U.S. Patent Application Ser. No.61/115,398, filed Nov. 17, 2008, solvent treatment (e.g., treatment withionic liquids) and freeze milling (see, e.g., U.S. Ser. No. 12/502,629).

Fermentation

Microorganisms can produce a number of useful intermediates andproducts, such as those described herein, by fermenting a low molecularweight sugar in the presence of the functionalized biomass materials.For example, fermentation or other bioprocesses can produce alcohols,organic acids, hydrocarbons, hydrogen, proteins or mixtures of any ofthese materials.

The microorganism can be a natural microorganism or an engineeredmicroorganism. For example, the microorganism can be a bacterium, e.g.,a cellulolytic bacterium, a fungus, e.g., a yeast, a plant or a protist,e.g., an algae, a protozoa or a fungus-like protist, e.g., a slime mold.When the organisms are compatible, mixtures of organisms can beutilized.

Suitable fermenting microorganisms have the ability to convertcarbohydrates, such as glucose, xylose, arabinose, mannose, galactose,oligosaccharides or polysaccharides into fermentation products.Fermenting microorganisms include strains of the genus Saccharomycesspp. e.g., Saccharomyces cerevisiae (baker's yeast), Saccharomycesdistaticus, Saccharomyces uvarum; the genus Kluyveromyces, e.g., speciesKluyveromyces marxianus, Kluyveromyces fragilis; the genus Candida,e.g., Candida pseudotropicalis, and Candida brassicae, Pichia stipitis(a relative of Candida shehatae, the genus Clavispora, e.g., speciesClavispora lusitaniae and Clavispora opuntiae, the genus Pachysolen,e.g., species Pachysolen tannophilus, the genus Bretannomyces, e.g.,species Bretannomyces clausenii (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212).

Commercially available yeasts include, for example, Red Star®/LesaffreEthanol Red (available from Red Star/Lesaffre, USA), FALI® (availablefrom Fleischmann's Yeast, a division of Burns Philip Food Inc., USA),SUPERSTART® (available from Alltech, now Lalemand), GERT STRAND®(available from Gert Strand AB, Sweden) and FERMOL® (available from DSMSpecialties).

Bacteria may also be used in fermentation, e.g., Zymomonas mobilis andClostridium thermocellum (Philippidis, 1996, supra).

The optimum pH for yeast is from about pH 4 to 5, while the optimum pHfor Zymomonas bacteria is from about pH 5 to 6. Typical fermentationtimes are about 24 to 96 hours with temperatures in the range of 26° C.to 40° C., however thermophilic microorganisms prefer highertemperatures.

In some embodiments, all or a portion of the fermentation process can beinterrupted before the low molecular weight sugar is completelyconverted to ethanol. The intermediate fermentation products includehigh concentrations of sugar and carbohydrates. These intermediatefermentation products can be used in preparation of food for human oranimal consumption. Additionally or alternatively, the intermediatefermentation products can be ground to a fine particle size in astainless-steel laboratory mill to produce a flour-like substance.

Mobile fermentors can be utilized, as described in U.S. ProvisionalPatent Application Ser. 60/832,735, now Published InternationalApplication No. WO 2008/011598.

Post-Processing

Distillation

After fermentation, the resulting fluids can be distilled using, forexample, a “beer column” to separate ethanol and other alcohols from themajority of water and residual solids. The vapor exiting the beer columncan be, e.g., 35% by weight ethanol and can be fed to a rectificationcolumn. A mixture of nearly azeotropic (92.5%) ethanol and water fromthe rectification column can be purified to pure (99.5%) ethanol usingvapor-phase molecular sieves. The beer column bottoms can be sent to thefirst effect of a three-effect evaporator. The rectification columnreflux condenser can provide heat for this first effect. After the firsteffect, solids can be separated using a centrifuge and dried in a rotarydryer. A portion (25%) of the centrifuge effluent can be recycled tofermentation and the rest sent to the second and third evaporatoreffects. Most of the evaporator condensate can be returned to theprocess as fairly clean condensate with a small portion split off towaste water treatment to prevent build-up of low-boiling compounds.

Intermediates and Products

The processes described herein can be used to produce one or moreintermediates or products, such as energy, fuels, foods and materials.Specific examples of products include, but are not limited to, hydrogen,alcohols (e.g., monohydric alcohols or dihydric alcohols, such asethanol, n-propanol or n-butanol), hydrated or hydrous alcohols, e.g.,containing greater than 10%, 20%, 30% or even greater than 40% water,xylitol, sugars, biodiesel, organic acids (e.g., acetic acid and/orlactic acid), hydrocarbons, co-products (e.g., proteins, such ascellulolytic proteins (enzymes) or single cell proteins), and mixturesof any of these in any combination or relative concentration, andoptionally in combination with any additives, e.g., fuel additives.Other examples include carboxylic acids, such as acetic acid or butyricacid, salts of a carboxylic acid, a mixture of carboxylic acids andsalts of carboxylic acids and esters of carboxylic acids (e.g., methyl,ethyl and n-propyl esters), ketones (e.g., acetone), aldehydes (e.g.,acetaldehyde), alpha, beta unsaturated acids, such as acrylic acid andolefins, such as ethylene. Other alcohols and alcohol derivativesinclude propanol, propylene glycol, 1,4-butanediol, 1,3-propanediol,methyl or ethyl esters of any of these alcohols. Other products includemethyl acrylate, methylmethacrylate, lactic acid, propionic acid,butyric acid, succinic acid, 3-hydroxypropionic acid, a salt of any ofthe acids and a mixture of any of the acids and respective salts.

Other intermediates and products, including food and pharmaceuticalproducts, are described in U.S. Ser. No. 12/417,900, the full disclosureof which is hereby incorporated by reference herein.

EXAMPLES

The following Examples are intended to illustrate, and do not limit theteachings of this disclosure.

Example 1 Preparation of Fibrous Material from Polycoated Paper

A 1500 pound skid of virgin, half-gallon juice cartons made ofun-printed polycoated white Kraft board having a bulk density of 20lb/ft³ was obtained from International Paper. Each carton was foldedflat, and then fed into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder was equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades was adjusted to 0.10inch. The output from the shredder resembled confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inchand a thickness equivalent to that of the starting material (about 0.075inch).

The confetti-like material was fed to a Munson rotary knife cutter,Model SC30. Model SC30 is equipped with four rotary blades, four fixedblades, and a discharge screen having ⅛ inch openings. The gap betweenthe rotary and fixed blades was set to approximately 0.020 inch. Therotary knife cutter sheared the confetti-like pieces across theknife-edges, tearing the pieces apart and releasing a fibrous materialat a rate of about one pound per hour. The fibrous material had a BETsurface area of 0.9748 m²/g+/−0.0167 m²/g, a porosity of 89.0437 percentand a bulk density (@0.53 psia) of 0.1260 g/mL. An average length of thefibers was 1.141 mm and an average width of the fibers was 0.027 mm,giving an average L/D of 42:1.

Example 2 Preparation of Fibrous Material from Bleached Kraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch and a thickness equivalent to that of the startingmaterial (about 0.075 inch). The confetti-like material was fed to aMunson rotary knife cutter, Model SC30. The discharge screen had ⅛ inchopenings. The gap between the rotary and fixed blades was set toapproximately 0.020 inch. The rotary knife cutter sheared theconfetti-like pieces, releasing a fibrous material at a rate of aboutone pound per hour. The fibrous material had a BET surface area of1.1316 m²/g+/−0.0103 m²/g, a porosity of 88.3285 percent and a bulkdensity (@0.53 psia) of 0.1497 g/mL. An average length of the fibers was1.063 mm and an average width of the fibers was 0.0245 mm, giving anaverage L/D of 43:1.

Example 3 Preparation of Twice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had 1/16 inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. The material resultingfrom the first shearing was fed back into the same setup described aboveand sheared again. The resulting fibrous material had a BET surface areaof 1.4408 m²/g+/−0.0156 m²/g, a porosity of 90.8998 percent and a bulkdensity (@0.53 psia) of 0.1298 g/mL. An average length of the fibers was0.891 mm and an average width of the fibers was 0.026 mm, giving anaverage L/D of 34:1.

Example 4 Preparation of Thrice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had ⅛ inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter sheared the confetti-like pieces across theknife-edges. The material resulting from the first shearing was fed backinto the same setup and the screen was replaced with a 1/16 inch screen.This material was sheared. The material resulting from the secondshearing was fed back into the same setup and the screen was replacedwith a 1/32 inch screen. This material was sheared. The resultingfibrous material had a BET surface area of 1.6897 m²/g+/−0.0155 m²/g, aporosity of 87.7163 percent and a bulk density (@0.53 psia) of 0.1448g/mL. An average length of the fibers was 0.824 mm and an average widthof the fibers was 0.0262 mm, giving an average L/D of 32:1.

Example 5 Electron Beam Processing

Samples were treated with electron beam using a vaulted Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 1 describes the parameters used. Table 2 reports thenominal dose used for the Sample ID (in Mrad) and the corresponding dosedelivered to the sample (in kgy).

TABLE 1 Rhodotron ® TT 200 Parameters Beam Beam Produced: Acceleratedelectrons Beam energy: Nominal (fixed): 10 MeV (+0 keV-250 keV Energydispersion at 10 Full width half maximum (FWHM) 300 keV Mev: Beam powerat 10 MeV: Guaranteed Operating Range 1 to 80 kW Power ConsumptionStand-by condition  <15 kW (vacuum and cooling ON): At 50 kW beam power:<210 kW At 80 kW beam power: <260 kW RF System Frequency: 107.5 ± 1 MHzTetrode type: Thomson TH781 Scanning Horn Nominal Scanning Length 120 cm(measured at 25-35 cm from window): Scanning Range: From 30% to 100% ofNominal Scanning Length Nominal Scanning 100 Hz ± 5% Frequency (at max.scanning length): Scanning Uniformity ±5% (across 90% of NominalScanning Length)

TABLE 2 Dosages Delivered to Samples Total Dosage (MRad) (NumberAssociated with Sample ID Delivered Dose (kgy)¹ 1 9.9 3 29.0 5 50.4 769.2 10 100.0 15 150.3 20 198.3 30 330.9 50 529.0 70 695.9 100 993.6¹For example, 9.9 kgy was delivered in 11 seconds at a beam current of 5mA and a line speed of 12.9 feet/minute. Cool time between treatmentswas around 2 minutes.

Example 6 Methods of Determining Molecular Weight of Cellulosic andLignocellulosic Materials by Gel Permeation Chromatography

Cellulosic and lignocellulosic materials for analysis were treatedaccording to Example 4. Sample materials presented in the followingtables include Kraft paper (P), wheat straw (WS), alfalfa (A), cellulose(C), switchgrass (SG), grasses (G), and starch (ST), and sucrose (S).The number “132” of the Sample ID refers to the particle size of thematerial after shearing through a 1/32 inch screen. The number after thedash refers to the dosage of radiation (MRad) and “US” refers toultrasonic treatment. For example, a sample ID “P132-10” refers to Kraftpaper that has been sheared to a particle size of 132 mesh and has beenirradiated with 10 Mrad.

For samples that were irradiated with e-beam, the number following thedash refers to the amount of energy delivered to the sample. Forexample, a sample ID “P-100e” refers to Kraft paper that has beendelivered a dose of energy of about 100 MRad or about 1000 kgy (Table2).

TABLE 3 Peak Average Molecular Weight of Irradiated Kraft Paper SampleSample Dosage¹ Average MW ± Source ID (MRad) Ultrasound² Std Dev. KraftPaper P132 0 No 32853 ± 10006 P132-10 10 ″  61398 ± 2468** P132-100 100″ 8444 ± 580  P132-181 181 ″ 6668 ± 77  P132-US 0 Yes 3095 ± 1013 **Lowdoses of radiation appear to increase the molecular weight of somematerials ¹Dosage Rate = 1 MRad/hour ²Treatment for 30 minutes with 20kHz ultrasound using a 1000 W horn under re-circulating conditions withthe material dispersed in water.

TABLE 4 Peak Average Molecular Weight of Irradiated Kraft Paper withE-Beam Sample Sample Dosage Average MW ± Source ID (MRad) Std Dev. KraftPaper P-1e 1 63489 ± 595 P-5e 5 56587 ± 536 P-10e 10 53610 ± 327 P-30e30 38231 ± 124 P-70e 70 12011 ± 158 P-100e 100 9770 ± 2 

TABLE 5 Peak Average Molecular Weight of Gamma Irradiated Materials PeakDosage¹ Average MW ± Sample ID # (MRad) Ultrasound² Std Dev. WS132 1  0No 1407411 ± 175191 2 ″ ″ 39145 ± 3425 3 ″ ″ 2886 ± 177 WS132-10* 1 10 ″26040 ± 3240 WS132-100* 1 100  ″ 23620 ± 453  A132 1  0 ″ 1604886 ±151701 2 ″ ″ 37525 ± 3751 3 ″ ″ 2853 ± 490 A132-10* 1 10 ″ 50853 ± 16652 ″ ″ 2461 ± 17  A132-100* 1 100  ″ 38291 ± 2235 2 ″ ″ 2487 ± 15  SG1321  0 ″ 1557360 ± 83693  2 ″ ″ 42594 ± 4414 3 ″ ″ 3268 ± 249 SG132-10* 110 ″ 60888 ± 9131 SG132-100* 1 100  ″ 22345 ± 3797 SG132-10-US 1 10 Yes 86086 ± 43518 2 ″ ″ 2247 ± 468 SG132-100-US 1 100  ″  4696 ± 1465*Peaks coalesce after treatment **Low doses of radiation appear toincrease the molecular weight of some materials ¹Dosage Rate = 1MRad/hour ²Treatment for 30 minutes with 20 kHz ultrasound using a 1000W horn under re-circulating conditions with the material dispersed inwater.

TABLE 6 Peak Average Molecular Weight of Irradiated Material with E-BeamPeak Average MW ± Sample ID # Dosage STD DEV. A-1e 1 1 1004783 ± 97518 234499 ± 482 3 2235 ± 1  A-5e 1 5 38245 ± 346 2 2286 ± 35 A-10e 1 1044326 ± 33  2 2333 ± 18 A-30e 1 30 47366 ± 583 2 2377 ± 7  A-50e 1 5032761 ± 168 2 2435 ± 6  G-1e 1 1  447362 ± 38817 2 32165 ± 779 3 3004 ±25 G-5e 1 5  62167 ± 6418 2 2444 ± 33 G-10e 1 10  72636 ± 4075 2 3065 ±34 G-30e 1 30 17159 ± 390 G-50e 1 50 18960 ± 142 ST 1 0 923336 ± 1883 2150265 ± 4033 ST-1e 1 1 846081 ± 5180 2 131222 ± 1687 ST-5e 1 5  90664 ±1370 ST-10e 1 10 98050 ± 255 ST-30e 1 30 41884 ± 223 ST-70e 1 70 9699 ±31 ST-100e 1 100 8705 ± 38

Gel Permeation Chromatography (GPC) is used to determine the molecularweight distribution of polymers. During GPC analysis, a solution of thepolymer sample is passed through a column packed with a porous geltrapping small molecules. The sample is separated based on molecularsize with larger molecules eluting sooner than smaller molecules. Theretention time of each component is most often detected by refractiveindex (RI), evaporative light scattering (ELS), or ultraviolet (UV) andcompared to a calibration curve. The resulting data is then used tocalculate the molecular weight distribution for the sample.

A distribution of molecular weights rather than a unique molecularweight is used to characterize synthetic polymers. To characterize thisdistribution, statistical averages are utilized. The most common ofthese averages are the “number average molecular weight” (M_(n)) and the“weight average molecular weight” (M_(w)).

Methods of calculating these values are described in the art, e.g., inExample 9 of WO 2008/073186.

The polydispersity index or PI is defined as the ratio of M_(w)/M_(n).The larger the PI, the broader or more disperse the distribution. Thelowest value that a PI can have is 1. This represents a monodispersesample; that is, a polymer with all of the molecules in the distributionbeing the same molecular weight.

The peak molecular weight value (M_(P)) is another descriptor defined asthe mode of the molecular weight distribution. It signifies themolecular weight that is most abundant in the distribution. This valuealso gives insight to the molecular weight distribution.

Most GPC measurements are made relative to a different polymer standard.The accuracy of the results depends on how closely the characteristicsof the polymer being analyzed match those of the standard used. Theexpected error in reproducibility between different series ofdeterminations, calibrated separately, is ca. 5-10% and ischaracteristic to the limited precision of GPC determinations.Therefore, GPC results are most useful when a comparison between themolecular weight distributions of different samples is made during thesame series of determinations.

The lignocellulosic samples required sample preparation prior to GPCanalysis. First, a saturated solution (8.4% by weight) of lithiumchloride (LiCl) was prepared in dimethyl acetamide (DMAc). Approximately100 mg of the sample was added to approximately 10 g of a freshlyprepared saturated LiCl/DMAc solution, and the mixture was heated toapproximately 150° C.-170° C. with stirring for 1 hour. The resultingsolutions were generally light- to dark-yellow in color. The temperatureof the solutions was decreased to approximately 100° C. and heated foran additional 2 hours. The temperatures of the solutions were thendecreased to approximately 50° C. and the sample solutions were heatedfor approximately 48 to 60 hours. Of note, samples irradiated at 100MRad were more easily solubilized as compared to their untreatedcounterpart. Additionally, the sheared samples (denoted by the number132) had slightly lower average molecular weights as compared with uncutsamples.

The resulting sample solutions were diluted 1:1 using DMAc as solventand were filtered through a 0.45 μm PTFE filter. The filtered samplesolutions were then analyzed by GPC using the parameters described inTable 7. The peak average molecular weights (Mp) of the samples, asdetermined by Gel Permeation Chromatography (GPC), are summarized inTables 3-6. Each sample was prepared in duplicate and each preparationof the sample was analyzed in duplicate (two injections) for a total offour injections per sample. The EasiCal® polystyrene standards PS1A andPS1B were used to generate a calibration curve for the molecular weightscale from about 580 to 7,500,00 Daltons.

TABLE 7 GPC Analysis Conditions Instrument: Waters Alliance GPC 2000Plgel 10μ Mixed-B Columns (3): S/N's: 10M-MB-148-83; 10M-MB-148-84;10M-MB-174-129 Mobile Phase (solvent): 0.5% LiCl in DMAc (1.0 mL/min.)Column/Detector Temperature: 70° C. Injector Temperature: 70° C. SampleLoop Size: 323.5 μL

Example 7 Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS)Surface Analysis

Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS) is asurface-sensitive spectroscopy that uses a pulsed ion beam (Cs ormicrofocused Ga) to remove molecules from the very outermost surface ofthe sample. The particles are removed from atomic monolayers on thesurface (secondary ions). These particles are then accelerated into a“flight tube” and their mass is determined by measuring the exact timeat which they reach the detector (i.e. time-of-flight). ToF-SIMSprovides detailed elemental and molecular information about the surface,thin layers, interfaces of the sample, and gives a fullthree-dimensional analysis. The use is widespread, includingsemiconductors, polymers, paint, coatings, glass, paper, metals,ceramics, biomaterials, pharmaceuticals and organic tissue. SinceToF-SIMS is a survey technique, all the elements in the periodic table,including H, are detected. ToF-SIMS data is presented in Tables 8-11.Parameters used are reported in Table 12.

TABLE 8 Normalized Mean Intensities of Various Positive Ions of Interest(Normalized relative to total ion counts × 10000) P132 P132-10 P132-100m/z species Mean σ Mean σ Mean σ 23 Na 257 28 276 54 193 36 27 Al 647 43821 399 297 44 28 Si 76 45.9 197 89 81.7 10.7 15 CH₃ 77.9 7.8 161 26 13312 27 C₂H₃ 448 28 720 65 718 82 39 C₃H₃ 333 10 463 37 474 26 41 C₃H₅ 70319 820 127 900 63 43 C₃H₇ 657 11 757 162 924 118 115 C₉H₇ 73 13.4 40.34.5 42.5 15.7 128 C₁₀H₈ 55.5 11.6 26.8 4.8 27.7 6.9 73 C₃H₉Si* 181 7765.1 18.4 81.7 7.5 147 C₅H₁₅OSi₂* 72.2 33.1 24.9 10.9 38.5 4 207C₅H₁₅O₃Si₃* 17.2 7.8 6.26 3.05 7.49 1.77 647 C₄₂H₆₄PO₃ 3.63 1.05 1.431.41 10.7 7.2

TABLE 9 Normalized Mean Intensities of Various Negative Ions of Interest(Normalized relative to total ion counts × 10000) P132 P132-10 P132-100m/z species Mean σ Mean σ Mean σ 19 F 15.3 2.1 42.4 37.8 19.2 1.9 35 Cl63.8 2.8 107 33 74.1 5.5 13 CH 1900 91 1970 26 1500 6 25 C₂H 247 127 22099 540 7 26 CN 18.1 2.1 48.6 30.8 43.9 1.4 42 CNO 1.16 0.71 0.743 0.71110.8 0.9 46 NO₂ 1.87 0.38 1.66 1.65 12.8 1.8

TABLE 10 Normalized Mean Intensities of Various Positive Ions ofInterest (Normalized relative to total ion counts × 10000) P-1e P-5eP-10e P-30e P-70e P-100e m/z Species Mean σ Mean σ Mean σ Mean σ Mean σMean σ 23 Na 232 56 370 37 241 44 518 57 350 27 542 104 27 Al 549 194677 86 752 371 761 158 516 159 622 166 28 Si 87.3 11.3 134 24 159 100158 32 93.7 17.1 124 11 15 CH₃ 114 23 92.9 3.9 128 18 110 16 147 16 1415 27 C₂H₃ 501 205 551 59 645 165 597 152 707 94 600 55 39 C₃H₃ 375 80288 8 379 82 321 57 435 61 417 32 41 C₃H₅ 716 123 610 24 727 182 607 93799 112 707 84 43 C₃H₇ 717 121 628 52 653 172 660 89 861 113 743 73 115C₉H₇ 49.9 14.6 43.8 2.6 42.2 7.9 41.4 10.1 27.7 8 32.4 10.5 128 C₁₀H₈38.8 13.1 39.2 1.9 35.2 11.8 31.9 7.8 21.2 6.1 24.2 6.8 73 C₃H₉Si* 92.53.0 80.6 2.9 72.3 7.7 75.3 11.4 63 3.4 55.8 2.1 147 C₅H₁₅OSi₂* 27.2 3.917.3 1.2 20.4 4.3 16.1 1.9 21.7 3.1 16.3 1.7 207 C₅H₁₅O₃Si₃* 6.05 0.743.71 0.18 4.51 0.55 3.54 0.37 5.31 0.59 4.08 0.28 647 C₄₂H₆₄PO₃ 1.611.65 1.09 1.30 0.325 0.307 nd ~ 0.868 1.31 0.306 0.334

TABLE 11 Normalized Mean Intensities of Various Negative Ions ofInterest (Normalized relative to total ion counts × 10000) P-1e P-5eP-10e P-30e P-70e P-100e m/z species Mean σ Mean σ Mean σ Mean σ Mean σMean σ 13 CH 1950 72 1700 65 1870 91 1880 35 2000 46 2120 102 25 C₂H 15447 98.8 36.3 157 4 230 17 239 22 224 19 19 F 25.4 1 24.3 1.4 74.3 18.640.6 14.9 25.6 1.9 21.5 2 35 Cl 39.2 13.5 38.7 3.5 46.7 5.4 67.6 6.245.1 2.9 32.9 10.2 26 CN 71.9 18.9 6.23 2.61 28.1 10.1 34.2 29.2 57.328.9 112 60 42 CNO 0.572 0.183 0.313 0.077 0.62 0.199 1.29 0.2 1.37 0.551.38 0.28 46 NO₂ 0.331 0.057 0.596 0.255 0.668 0.149 1.44 0.19 1.92 0.290.549 0.1

TABLE 12 ToF-SIMS Parameters Instrument Conditions: Instrument: PHITRIFT II Primary Ion Source: ⁶⁹Ga Primary Ion Beam Potential: 12 kV +ions 18 kV − ions Primary Ion Current (DC): 2 na for P#E samples 600 pAfor P132 samples Energy Filter/CD: Out/Out Masses Blanked: None ChargeCompensation: On

ToF-SIMS uses a focused, pulsed particle beam (typically Cs or Ga) todislodge chemical species on a materials surface. Particles producedcloser to the site of impact tend to be dissociated ions (positive ornegative). Secondary particles generated farther from the impact sitetend to be molecular compounds, typically fragments of much largerorganic macromolecules. The particles are then accelerated into a flightpath on their way towards a detector. Because it is possible to measurethe “time-of-flight” of the particles from the time of impact todetector on a scale of nano-seconds, it is possible to produce a massresolution as fine as 0.00× atomic mass units (i.e. one part in athousand of the mass of a proton). Under typical operating conditions,the results of ToF-SIMS analysis include: a mass spectrum that surveysall atomic masses over a range of 0-10,000 amu, the rastered beamproduces maps of any mass of interest on a sub-micron scale, and depthprofiles are produced by removal of surface layers by sputtering underthe ion beam. Negative ion analysis showed that the polymer hadincreasing amounts of CNO, CN, and NO₂ groups.

Example 8 Porosimetry Analysis of Irradiated Materials

Mercury pore size and pore volume analysis (Table 21) is based onforcing mercury (a non-wetting liquid) into a porous structure undertightly controlled pressures. Since mercury does not wet most substancesand will not spontaneously penetrate pores by capillary action, it mustbe forced into the voids of the sample by applying external pressure.The pressure required to fill the voids is inversely proportional to thesize of the pores. Only a small amount of force or pressure is requiredto fill large voids, whereas much greater pressure is required to fillvoids of very small pores.

TABLE 21 Pore Size and Volume Distribution by Mercury Porosimetry MedianMedian Average Bulk Total Total Pore Pore Pore Density ApparentIntrusion Pore Diameter Diameter Diameter @ 0.50 (skeletal) Volume Area(Volume) (Area) (4V/A) psia Density Porosity Sample ID (mL/g) (m²/g)(μm) (μm) (μm) (g/mL) (g/mL) (%) P132 6.0594 1.228 36.2250 13.727819.7415 0.1448 1.1785 87.7163 P132-10 5.5436 1.211 46.3463 4.564618.3106 0.1614 1.5355 89.4875 P132-100 5.3985 0.998 34.5235 18.200521.6422 0.1612 1.2413 87.0151 P132-181 3.2866 0.868 25.3448 12.241015.1509 0.2497 1.3916 82.0577 P132-US 6.0005 14.787 98.3459 0.00551.6231 0.1404 0.8894 84.2199 A132 2.0037 11.759 64.6308 0.0113 0.68160.3683 1.4058 73.7990 A132-10 1.9514 10.326 53.2706 0.0105 0.7560 0.37681.4231 73.5241 A132-100 1.9394 10.205 43.8966 0.0118 0.7602 0.37601.3889 72.9264 SG132 2.5267 8.265 57.6958 0.0141 1.2229 0.3119 1.470878.7961 5G132-10 2.1414 8.643 26.4666 0.0103 0.9910 0.3457 1.331574.0340 5G132-100 2.5142 10.766 32.7118 0.0098 0.9342 0.3077 1.359077.3593 5G132-10-US 4.4043 1.722 71.5734 1.1016 10.2319 0.1930 1.288385.0169 5G132-100-US 4.9665 7.358 24.8462 0.0089 2.6998 0.1695 1.073184.2010 WS132 2.9920 5.447 76.3675 0.0516 2.1971 0.2773 1.6279 82.9664WS132-10 3.1138 2.901 57.4727 0.3630 4.2940 0.2763 1.9808 86.0484WS132-100 3.2077 3.114 52.3284 0.2876 4.1199 0.2599 1.5611 83.3538 A-1e1.9535 3.698 25.3411 0.0810 2.1130 0.3896 1.6299 76.0992 A-5e 1.96976.503 29.5954 0.0336 1.2117 0.3748 1.4317 73.8225 A-10e 2.0897 12.03045.5493 0.0101 0.6948 0.3587 1.4321 74.9545 A-50e 2.1141 7.291 37.07600.0304 1.1599 0.3577 1.4677 75.6264 G-1e 2.4382 7.582 58.5521 0.02011.2863 0.3144 1.3472 76.6610 G-5e 2.4268 6.436 44.4848 0.0225 1.50820.3172 1.3782 76.9831 G-10e 2.6708 6.865 62.8605 0.0404 1.5562 0.29601.4140 79.0638 G-50e 2.8197 6.798 56.5048 0.0315 1.6591 0.2794 1.317978.7959 P-1e 7.7692 1.052 49.8844 22.9315 29.5348 0.1188 1.5443 92.3065P-5e 7.1261 1.212 46.6400 12.3252 23.5166 0.1268 1.3160 90.3644 P-10e6.6096 1.113 41.4252 17.4375 23.7513 0.1374 1.4906 90.7850 P-50e 6.59111.156 40.7837 15.9823 22.7974 0.1362 1.3302 89.7616 P-100e 5.3507 1.19535.3622 10.7400 17.9063 0.1648 1.3948 88.1840 S 0.4362 0.030 102.841142.5047 57.8208 0.9334 1.5745 40.7160 S-1e 0.3900 0.632 90.6808 0.00412.4680 0.9772 1.5790 38.1140 S-5e 0.3914 0.337 97.1991 0.0070 4.64060.9858 1.6052 38.5847 S-10e 0.4179 0.349 113.4360 0.0042 4.7873 0.94691.5669 39.5678 S-30e 0.4616 5.329 102.0559 0.0042 0.3464 0.9065 1.558541.8388 S-50e 0.5217 7.162 137.2124 0.0051 0.2914 0.8521 1.5342 44.4582S-100e 0.8817 15.217 76.4577 0.0053 0.2318 0.6478 1.5105 57.1131 St0.6593 17.631 4.2402 0.0053 0.1496 0.7757 1.5877 51.1438 St-1e 0.672018.078 4.3360 0.0052 0.1487 0.7651 1.5750 51.4206 St-5e 0.6334 19.4954.2848 0.0051 0.1300 0.7794 1.5395 49.3706 St-10e 0.6208 16.980 4.33620.0056 0.1462 0.7952 1.5703 49.3630 St-30e 0.6892 18.066 4.4152 0.00500.1526 0.7475 1.5417 51.5165 St-50e 0.6662 18.338 4.3759 0.0054 0.14530.7637 1.5548 50.8778 St-100e 0.6471 23.154 5.4032 0.0048 0.1118 0.72291.3582 46.7761

The AutoPore 9520 can attain a maximum pressure of 414 MPa or 60,000psia. There are four low pressure stations for sample preparation andcollection of macropore data from 0.2 psia to 50 psia. There are twohigh pressure chambers, which collect data from 25 psia to 60,000 psia.The sample is placed in a bowl-like apparatus called a penetrometer,which is bonded to a glass capillary stem with a metal coating. Asmercury invades the voids in and around the sample, it moves down thecapillary stem. The loss of mercury from the capillary stem results in achange in the electrical capacitance. The change in capacitance duringthe experiment is converted to volume of mercury by knowing the stemvolume of the penetrometer in use. A variety of penetrometers withdifferent bowl (sample) sizes and capillaries are available toaccommodate most sample sizes and configurations. Table 22 below definessome of the key parameters calculated for each sample.

TABLE 22 Definition of Parameters Parameter Description Total IntrusionVolume: The total volume of mercury intruded during an experiment. Thiscan include interstitial filling between small particles, porosity ofsample, and compression volume of sample. Total Pore Area: The totalintrusion volume converted to an area assuming cylindrical shaped pores.Median Pore Diameter (volume): The size at the 50^(th) percentile on thecumulative volume graph. Median Pore Diameter (area): The size at the50^(th) percentile on the cumulative area graph. Average Pore Diameter:The total pore volume divided by the total pore area (4 V/A). BulkDensity: The mass of the sample divided by the bulk volume. Bulk volumeis determined at the filling pressure, typically 0.5 psia. ApparentDensity: The mass of sample divided by the volume of sample measured athighest pressure, typically 60,000 psia. Porosity: (BulkDensity/Apparent Density) × 100%

Example 9 Particle Size Analysis of Irradiated Materials

The technique of particle sizing by static light scattering is based onMie theory (which also encompasses Fraunhofer theory). Mie theorypredicts the intensity vs. angle relationship as a function of the sizefor spherical scattering particles provided that other system variablesare known and held constant. These variables are the wavelength ofincident light and the relative refractive index of the sample material.Application of Mie theory provides the detailed particle sizeinformation. Table 23 summarizes particle size using median diameter,mean diameter, and modal diameter as parameters.

TABLE 23 Particle Size by Laser Light Scattering (Dry Sample Dispersion)Median Mean Modal Sample ID Diameter (μm) Diameter (μm) Diameter (μm)A132 380.695 418.778 442.258 A132-10 321.742 366.231 410.156 A132-100301.786 348.633 444.169 SG132 369.400 411.790 455.508 SG132-10 278.793325.497 426.717 SG132-100 242.757 298.686 390.097 WS132 407.335 445.618467.978 WS132-10 194.237 226.604 297.941 WS132-100 201.975 236.037307.304

Particle size was determined by Laser Light Scattering (Dry SampleDispersion) using a Malvern Mastersizer 2000 using the followingconditions:

-   -   Feed Rate: 35%    -   Disperser Pressure: 4 Bar    -   Optical Model: (2.610, 1.000i), 1.000

An appropriate amount of sample was introduced onto a vibratory tray.The feed rate and air pressure were adjusted to ensure that theparticles were properly dispersed. The key component is selecting an airpressure that will break up agglomerations, but does not compromise thesample integrity. The amount of sample needed varies depending on thesize of the particles. In general, samples with fine particles requireless material than samples with coarse particles.

Example 10 Surface Area Analysis of Irradiated Materials

Surface area of each sample was analyzed using a Micromeritics ASAP 2420Accelerated Surface Area and Porosimetry System. The samples wereprepared by first degassing for 16 hours at 40° C. Next, free space(both warm and cold) with helium is calculated and then the sample tubeis evacuated again to remove the helium. Data collection begins afterthis second evacuation and consists of defining target pressures, whichcontrol how much gas is dosed onto the sample. At each target pressure,the quantity of gas adsorbed and the actual pressure are determined andrecorded. The pressure inside the sample tube is measured with apressure transducer. Additional doses of gas will continue until thetarget pressure is achieved and allowed to equilibrate. The quantity ofgas adsorbed is determined by summing multiple doses onto the sample.The pressure and quantity define a gas adsorption isotherm and are usedto calculate a number of parameters, including BET surface area (Table24).

TABLE 24 Summary of Surface Area by Gas Adsorption BET Surface Sample IDSingle point surface area (m²/g) Area (m²/g) P132 @ P/Po = 0.2503877711.5253 1.6897 P132-10 @ P/Po = 0.239496722 1.0212 1.2782 P132-100 @ P/Po= 0.240538100 1.0338 1.2622 P132-181 @ P/Po = 0.239166091 0.5102 0.6448P132-US @ P/Po = 0.217359072 1.0983 1.6793 A132 @ P/Po = 0.2400406100.5400 0.7614 A132-10 @ P/Po = 0.211218936 0.5383 0.7212 A132-100 @ P/Po= 0.238791097 0.4258 0.5538 SG132 @ P/Po = 0.237989353 0.6359 0.8350SG132-10 @ P/Po = 0.238576905 0.6794 0.8689 SG132-100 @ P/Po =0.241960361 0.5518 0.7034 SG132-10-US @ P/Po = 0.225692889 0.5693 0.7510SG132-100-US @ P/Po = 0.225935246 1.0983 1.4963 G-10-US 0.751 G100-US1.496 G132-US 1.679 WS132 @ P/Po = 0.237823664 0.6582 0.8663 WS132-10 @P/Po = 0.238612476 0.6191 0.7912 WS132-100 @ P/Po = 0.238398832 0.62550.8143 A-1e @ P/Po = 0.238098138 0.6518 0.8368 A-5e @ P/Po = 0.2431844770.6263 0.7865 A-10e @ P/Po = 0.243163236 0.4899 0.6170 A-50e @ P/Po =0.243225512 0.4489 0.5730 G-1e @ P/Po = 0.238496102 0.5489 0.7038 G-5e @P/Po = 0.242792602 0.5621 0.7086 G-10e @ P/Po = 0.243066031 0.50210.6363 G-50e @ P/Po = 0.238291132 0.4913 0.6333 P-1e @ P/Po =0.240842223 1.1413 1.4442 P-5e @ P/Po = 0.240789274 1.0187 1.3288 P-10e@ P/Po = 0.240116967 1.1015 1.3657 P-50e @ P/Po = 0.240072114 1.00891.2593 P-100e @ P/Po = 0.236541386 0.9116 1.1677 S @ P/Po = 0.2253350380.0147 0.0279 S-1e @ P/Po = 0.217142291 0.0193 0.0372 S-5e @ P/Po =0.133107838 0.0201 0.0485 S-10e @ P/Po = 0.244886517 0.0236 0.0317 S-30e@ P/Po = 0.237929400 0.0309 0.0428 S-50e @ P/Po = 0.245494494 0.02620.0365 S-100e @ P/Po = 0.224698551 0.0368 0.0506 St @ P/Po = 0.2383249490.3126 0.4013 St-1e @ P/Po = 0.238432726 0.3254 0.4223 St-5e @ P/Po =0.238363587 0.3106 0.4071 St-10e @ P/Po = 0.238341099 0.3205 0.4268St-30e @ P/Po = 0.238629889 0.3118 0.4189 St-50e @ P/Po = 0.2446309800.3119 0.3969 St-100e @ P/Po = 0.238421621 0.2932 0.3677

The BET model for isotherms is a widely used theory for calculating thespecific surface area. The analysis involves determining the monolayercapacity of the sample surface by calculating the amount required tocover the entire surface with a single densely packed layer of krypton.The monolayer capacity is multiplied by the cross sectional area of amolecule of probe gas to determine the total surface area. Specificsurface area is the surface area of the sample aliquot divided by themass of the sample.

Example 11 Fiber Length Determination of Irradiated Materials

Fiber length distribution testing was performed in triplicate on thesamples submitted using the Techpap MorFi LB01 system. The average fiberlength and width are reported in Table 25.

TABLE 25 Summary of Lignocellulosic Fiber Length and Width Data Arith-Average Statistically Width metic Length Corrected Average (microm-Average Weighted in Length Weighted eters) Sample ID (mm) Length (mm) inLength (mm) (μm) P132-10 0.484 0.615 0.773 24.7 P132-100 0.369 0.4230.496 23.8 P132-181 0.312 0.342 0.392 24.4 A132-10 0.382 0.423 0.65043.2 A132-100 0.362 0.435 0.592 29.9 SG132-10 0.328 0.363 0.521 44.0SG132-100 0.325 0.351 0.466 43.8 WS132-10 0.353 0.381 0.565 44.7WS132-100 0.354 0.371 0.536 45.4

Example 12 Fourier Transform Infrared (FT-IR) Spectrum of Irradiated andUnirradiated Kraft Paper

FT-IR analysis was performed on a Nicolet/Impact 400. The resultsindicate that samples P132, P132-10, P132-100, P-1e, P-5e, P-10e, P-30e,P-70e, and P-100e are consistent with a cellulose-based material.

FIG. 3 is an infrared spectrum of Kraft board paper sheared according toExample 4, while FIG. 4 is an infrared spectrum of the Kraft paper ofFIG. 3 after irradiation with 100 Mrad of gamma radiation. Theirradiated sample shows an additional peak in region A (centered about1730 cm⁻¹) that is not found in the un-irradiated material. Of note, anincrease in the amount of a carbonyl absorption at ˜1650 cm⁻¹ wasdetected when going from P132 to P132-10 to P132-100. Similar resultswere observed for the samples P-1e, P-5e, P-10e, P-30e, P-70e, andP-100e.

Example 13 Proton and Carbon-13 Nuclear Magnetic Resonance (¹H-NMR and¹³C-NMR) Spectra of Irradiated and Unirradiated Kraft Paper

Sample Preparation

The samples P132, P132-10, P132-100, P-1e, P-5e, P-10e, P-30e, P-70e,and P-100e were prepared for analysis by dissolution with DMSO-d₆ with2% tetrabutyl ammonium fluoride trihydrate. The samples that hadundergone lower levels of irradiation were significantly less solublethan the samples with higher irradiation. Unirradiated samples formed agel in this solvent mixture, but heating to 60° C. resolved the peaks inthe NMR spectra. The samples having undergone higher levels ofirradiation were soluble at a concentration of 10% wt/wt.

Analysis

¹H-NMR spectra of the samples at 15 mg/mL showed a distinct very broadresonance peak centered at 16 ppm (FIGS. 5A-5J). This peak ischaracteristic of an exchangeable —OH proton for an enol and wasconfirmed by a “d₂O shake.” Model compounds (acetylacetone, glucuronicacid, and keto-gulonic acid) were analyzed and made a convincing casethat this peak was indeed an exchangeable enol proton. This proposedenol peak was very sensitive to concentration effects, and we wereunable to conclude whether this resonance was due to an enol or possiblya carboxylic acid.

The carboxylic acid proton resonances of the model compounds weresimilar to what was observed for the treated cellulose samples. Thesemodel compounds were shifted up field to ˜5-6 ppm. Preparation of P-100eat higher concentrations (˜10% wt/wt) led to the dramatic down fieldshifting to where the carboxylic acid resonances of the model compoundswere found (˜6 ppm) (FIG. 5N). These results lead to the conclusion thatthis resonance is unreliable for characterizing this functional group,however the data suggests that the number of exchangeable hydrogensincreases with increasing irradiation of the sample. Also, no vinylprotons were detected.

The ¹³C NMR spectra of the samples confirm the presence of a carbonyl ofa carboxylic acid or a carboxylic acid derivative. This new peak (at 168ppm) is not present in the untreated samples (FIG. 5K). A ¹³C NMRspectrum with a long delay allowed the quantitation of the signal forP-100e (FIGS. 5L-5M). Comparison of the integration of the carbonylresonance to the resonances at approximately 100 ppm (the C1 signals)suggests that the ratio of the carbonyl carbon to C1 is 1:13.8 orroughly 1 carbonyl for every 14 glucose units. The chemical shift at 100ppm correlates well with glucuronic acid.

Titration

Samples P-100e and P132-100 (1 g) were suspended in deionized water (25mL). The indicator alizarin yellow was added to each sample withstirring. P-100e was more difficult to wet. Both samples were titratedwith a solution of 0.2M NaOH. The end point was very subtle and wasconfirmed by using pH paper. The starting pH of the samples was ˜4 forboth samples. P132-100 required 0.4 milliequivalents of hydroxide, whichgives a molecular weight for the carboxylic acid of 2500 amu. If 180 amuis used for a monomer, this suggests there is one carboxylic acid groupfor 13.9 monomer units. Likewise, P-100e required 3.2 milliequivalentsof hydroxide, which calculates to be one carboxylic acid group for every17.4 monomer units.

Conclusions

The C-6 carbon of cellulose appears to be oxidized to the carboxylicacid (a glucuronic acid derivative) in this oxidation is surprisinglyspecific. This oxidation is in agreement with the IR band that growswith irradiation at ˜1740 cm⁻¹, which corresponds to an aliphaticcarboxylic acid. The titration results are in agreement with thequantitative ¹³C NMR. The increased solubility of the sample with thehigher levels of irradiation correlates well with the increasing numberof carboxylic acid protons. A proposed mechanism for the degradation of“C-6 oxidized cellulose” is provided below in Scheme 1.

Example 14 Microbial Testing of Pretreated Biomass

Specific lignocellulosic materials pretreated as described herein areanalyzed for toxicity to common strains of yeast and bacteria used inthe biofuels industry for the fermentation step in ethanol production.Additionally, sugar content and compatibility with cellulase enzymes areexamined to determine the viability of the treatment process. Testing ofthe pretreated materials is carried out in two phases as follows.

Phase 1: Toxicity and Sugar Content

Toxicity of the pretreated grasses and paper feedstocks is measured inyeast Saccharomyces cerevisiae (wine yeast) and Pichia stipitis (ATCC66278) as well as the bacteria Zymomonas mobilis (ATCC 31821) andClostridium thermocellum (ATCC 31924). A growth study is performed witheach of the organisms to determine the optimal time of incubation andsampling.

Each of the feedstocks is then incubated, in duplicate, with S.cerevisiae, P. stipitis, Z. mobilis, and C. thermocellum in a standardmicrobiological medium for each organism. YM broth is used for the twoyeast strains, S. cerevisiae and P. stipitis. RM medium is used for Z.mobilis and CM4 medium for C. thermocellum. A positive control, withpure sugar added, but no feedstock, is used for comparison. During theincubation, a total of five samples is taken over a 12 hour period attime 0, 3, 6, 9, and 12 hours and analyzed for viability (plate countsfor Z. mobilis and direct counts for S. cerevisiae) and ethanolconcentration.

Sugar content of the feedstocks is measured using High PerformanceLiquid Chromatography (HPLC) equipped with either a Shodex™ sugar SP0810or Biorad Aminex® HPX-87P column. Each of the feedstocks (approx. 5 g)is mixed with reverse osmosis (RO) water for 1 hour. The liquid portionof the mixture is removed and analyzed for glucose, galactose, xylose,mannose, arabinose, and cellobiose content. The analysis is performedaccording to National Bioenergy Center protocol Determination ofStructural Carbohydrates and Lignin in Biomass.

Phase 2: Cellulase Compatibility

Feedstocks are tested, in duplicate, with commercially availableAccellerase® 1000, which contains a complex of enzymes that reduceslignocellulosic biomass into fermentable sugars, at the recommendedtemperature and concentration in an Erlenmeyer flask. The flasks areincubated with moderate shaking at around 200 rpm for 12 hours. Duringthat time, samples are taken every three hours at time 0, 3, 6, 9, and12 hours to determine the concentration of reducing sugars (Hope andDean, Biotech J., 1974, 144:403) in the liquid portion of the flasks.

Example 15 Sugar Concentration Analysis Using HPLC

13 samples were analyzed for sugar concentration (HPLC) and toxicityagainst 3 microorganisms (Pichia stipitis, Saccharomyces cerevisiae, andZymomonas mobilis. Table 26 lists the equipment used for theseexperiments. Table 27 and 28 provide a list of the sugars (includingvendor and lot numbers) used to prepare the HPLC standard and theprotocol used to prepare the HPLC standard, respectively.

TABLE 26 Equipment Utilized in Experiments Equipment Manufacturer, NamepH meter Orion Shakers (2) B. Braun Biotech, Certomat BS-1 HPLC Waters,2690 HPLC Module Spectrophotometer Unicam, UV300 YSI Biochem AnalyzerInterscience, YSI

TABLE 27 Sugars used in HPLC analysis Sugar Manufacturer Ref # Lot #glucose BioChemika 49140 1284892 xylose 95731 1304473 51707231cellobiose 22150 1303157 14806191 arabinose 10840 1188979 24105272mannose 63582 363063/1 22097 galactose 48259 46032/1 33197

TABLE 28 Preparation of HPLC standards Desired Volume of TotalConcentration Volume of sugar Nanopure Water Volume (mg/mL) solution(mL) (mL) 4 50 mL of 4 mg/mL 0 50 2 25 mL of 4 mg/mL 25 50 1 25 mL of 2mg/mL 25 50 0.5 25 mL of 1 mg/mL 25 50 0.1 5 ml of 1 mg/mL 20 25Verification 18.75 mL of 4 mg/mL 31.25 50 Standard 1.5 mg/mLAnalysis

Each sample (1 gram) was mixed with reverse osmosis water at 200 rpm and50° C. overnight. The pH of the sample was adjusted to between 5 and 6and filtered through a 0.2 μm syringe filter. Samples were stored at−20° C. prior to analysis to maintain integrity of the samples. Theobservations made during the preparation of the samples are presented inTable 29.

TABLE 29 Observations During HPLC Sample Preparation Amount Water usedadded Sample (g) (mL) pH Observations P132 1 30 5.38 Fluffy, difficultto mix P132-10 1 25 6.77 Fluffy, difficult to mix P132-100 1 20 3.19 pHis low, difficult to bring to pH 5.0, used 10N NaOH P132-US 0.3 5 6.14A132 1 15 5.52 A132-10 1 15 4.9 A132-100 1 15 5.39 SG132 1 15 5.59SG132-10 1 15 5.16 SG132-100 1 15 4.7 SG132-10-US 0.3 5 5.12SG132-100-US 0.3 5 4.97 WS132 1 15 5.63 WS132-10 1 15 5.43 WS132-100 115 5.02 *pH of these samples was adjusted to pH using 1N NaOH

Standards were prepared fresh from a 4 mg/mL stock solution of the 6combined sugars, glucose, xylose, cellobiose, arabinose, mannose, andgalactose. The stock solution was prepared by dissolving 0.400 grams ofeach sugar into 75 mL of nanopure water (0.3 micron filtered). Oncedissolved, the stock solution was diluted to 100 mL using a volumetricflask and stored at −20° C. Working standard solutions of 0.1, 0.5, 1,2, and 4 mg/mL were prepared by serial dilution of the stock solutionwith nanopure water. In addition, a verification standard of 1.5 mg/mLwas also prepared from the stock solution.

Sugar concentrations were analyzed according to the protocolDetermination of Structural Carbohydrates in Biomass (NREL BiomassProgram, 2006) and this protocol is incorporated herein by reference inits entirety. A SHODEX SUGAR SP0810 COLUMN with an Evaporative LightScattering Detector was used. A verification standard (1.5 mg/mL ofstandard) was analyzed every 8 injections to ensure that the integrityof the column and detector were maintained during the experiment. Thestandard curve coefficient of variation (R² value) was at least 0.989and the concentration of the verification standards were within 10% ofthe actual concentration. The HPLC conditions were as follows:

TABLE 30 HPLC Parameters Injection volume: 20 μL Mobile phase: nanopurewater*, 0.45 μm filtered and degassed Flow rate: 0.5 mL/min Columntemperature: 85° C. Detector temperature: evaporator temperature 110°C., nebulizer temperature 90° C. *Initial tests noted that betterseparation was observed when using nanopure water than 15/85acetonitrile:water in the mobile phase (manufacturer does not recommendusing greater than 20% acetonitrile with this column).Results

The results of the HPLC analysis are presented in Tables 31, 32, and 33.

TABLE 31 Sugar Concentration Expressed as mg/mL and mg/g of ExtractXylose Arabinose Glucose Cellobiose mW ~ 150 mW ~ 150 mW ~ 180 GalactoseMannose mW ~ 342 C₅H₁₀O₅ C₅H₁₀O₅ C₆H₁₂O₆ (see gluc) (see gluc) C₁₂H₂₂O₁₁Mono Mono Mono mg/mL:mg/g mg/mL:mg/g Disacc mg/ mg/ mg/ mg/ mg/ mg/Sample ID mL mg/g mL mg/g mL mg/g mL mg/g mL mg/g mL mg/g P P-132 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 P-132-10 0.000.00 0.00 0.00 0.34 8.60 0.00 0.00 0.00 0.00 00.33 8.13 P-132-100 0.357.04 0.00 0.00 0.34 6.14 0.00 0.00 0.00 0.00 0.36 7.20 P-132-BR 0.355.80 0.43 7.17 0.34 5.62 0.00 0.00 0.00 0.00 0.00 0.00 G G-132 0.39 5.880.38 5.73 0.84 12.66 0.34 5.04 0.92 13.76 0.00 0.00 G-132-10 0.50 7.500.41 6.18 1.07 16.04 0.35 5.19 0.98 14.66 0.00 0.00 G-132-100 0.00 0.000.37 5.54 0.41 6.14 0.00 0.00 0.55 8.28 0.45 6.71 G-132-10-US 0.34 5.730.39 6.45 0.33 5.43 0.00 0.00 0.00 0.00 0.00 0.00 G-132-100-US 0.00 0.000.37 6.22 0.35 5.90 0.33 5.43 0.40 6.70 0.39 6.45 A A-132 1.36 20.390.00 0.00 1.08 16.22 0.39 5.84 1.07 16.02 0.00 0.00 A-132-10 1.19 17.870.00 0.00 0.00 0.00 0.00 0.00 0.37 5.52 0.00 0.00 A-132-100 1.07 16.110.00 0.00 0.35 5.18 0.00 0.00 0.00 0.00 0.81 12.2 WS WS-132 0.49 7.410.41 6.15 0.39 5.90 0.00 0.00 0.00 0.00 0.00 0.00 WS-132-10 0.57 8.490.40 5.99 0.73 10.95 0.34 5.07 0.50 7.55 0.00 0.00 WS-132-100 0.43 6.390.37 5.51 0.36 5.36 0.00 0.00 0.36 5.33 0.35 5.25

TABLE 32 Sugar Concentration Expressed at % of Paper Sugar concentra-tion (% of dry sample) P132 P132-10 P132-100 P132-US cellobiose 0.000.81 0.72 0.00 glucose 0.00 0.86 0.67 0.56 xylose 0.00 0.00 0.70 0.58galactose 0.00 0.00 0.00 0.00 arabinose 0.00 0.00 0.00 0.72 mannose 0.000.00 0.00 0.00

TABLE 33 Sugar Concentration Expressed at % of Total Sample Sugarconcentration (% of dry A132- A132- SG132- SG132- SG132- SG132- WS132-WS132- sample) A132 10 100 SG132 10 100 10-US 100-US WS132 10 100Cellobiose 0.00 0.00 1.22 0.00 0.00 0.67 0.00 0.65 0.00 0.00 0.53Glucose 1.62 0.00 0.52 1.27 1.60 0.61 0.54 0.59 0.59 1.10 0.54 Xylose2.04 1.79 1.61 0.59 0.75 0.00 0.57 0.00 0.74 0.85 0.64 Galactose 0.580.00 0.00 0.50 0.52 0.00 0.00 0.54 0.00 0.51 0.00 Arabinose 0.00 0.000.00 0.57 0.62 0.55 0.65 0.62 0.62 0.60 0.55 Mannose 1.60 0.55 0.00 1.381.47 0.83 0.00 0.67 0.00 0.76 0.53

Example 16 Toxicity Study

Twelve samples were analyzed for toxicity against a panel of threeethanol-producing cultures. In this study, glucose was added to thesamples in order to distinguish between starvation of the cultures andtoxicity of the samples. A thirteenth sample was tested for toxicityagainst Pichia stipitis. A summary of the protocol used is listed inTable 32. A description of the chemicals and equipment used in thetoxicity testing is reported in Tables 34-36.

TABLE 34 Conditions for Toxicity Testing Organism ZymomonasSaccharomyces Pichia mobilis cerevesiae stipitis ATCC ATCC NRRL Variable31821 24858 Y-7124 Test Repetition Duplicate Inoculation  1   0.1  1Volume (mL) Incubation 30° C. 25° C. 25° C. Temperature Shaker Speed(rpm) 125 200  125 Erlenmeyer 250 mL 500 mL 250 mL Flask Volume Mediavolume 100 mL 100 mL 100 mL Total Incubation  36 36  48 time (hours)Ethanol Analysis 24, 30, 36 24, 30, 36 24, 36, 48 (hours) Cell Counts24, 36 24, 36 24, 48 (hours) pH 0 hours 0 hours 0 hours

TABLE 35 Reagents Used for Toxicity Testing Media Component ManufacturerReference # Lot # Urea ScholAR Chemistry 9472706 AD-7284-43 YeastNitrogen Base Becton Dickinson 291940 7128171 Peptone Becton Dickinson211677 4303198 Xylose Fluka 95731 1304473 51707231 Glucose Sigma G-5400107H0245 Yeast Extract Becton Dickinson 288620 4026828 (used for S.cerevisiae) Yeast Extract (used Becton Dickinson 212750 7165593 for P.stipitis and Z. mobilis) MgSO₄•7H₂O Sigma M5921 034K0066 (NH₄)₂SO₄ SigmaA4418 117K5421 KH₂PO₄ Sigma P5379 074K0160 YM Broth Becton Dickinson271120 6278265

TABLE 36 YSI Components Used in Shake Flask Study Component Catalog #Lot # YSI Ethanol Membrane 2786 07L100153 YSI Ethanol Standard (3.2 g/L)2790 012711040 YSI Ethanol Buffer 2787 07M1000053, 07100215

Testing was performed using the three microorganisms as described below.

Saccharomyces cerevisiae ATCC 24858 (American Type Culture Collection)

A slant of S. cerevisiae was prepared from a rehydrated lyophilizedculture obtained from ATCC. A portion of the slant material was streakedonto an YM Broth+20 g/L agar (pH 5.0) and incubated at 30° C. for 2days. A 250 mL Erlenmeyer flask containing 50 mL of medium (20 g/Lglucose, 3 g/L yeast extract, and 5.0 g/L peptone, pH 5.0) wasinoculated with one colony from the YM plate and incubated for 24 hoursat 25° C. and 200 rpm. After 23 hours of growth, a sample was taken andanalyzed for optical density (600 nm in a UV spectrophotometer) andpurity (Gram stain). Based on these results, one flask (called the SeedFlask) with an OD of 14.8 and clean Gram Stain was chosen to inoculateall of the test flasks.

The test vessels were 500 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved at 121° C.and 15 psi prior to the addition of the test materials. The testmaterials were not sterilized, as autoclaving will change the content ofthe samples. The test samples were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 1 mL (1% v/v) of seed flask material wasadded to each flask. The flasks were incubated as described above for 36hours.

Pichia stipitis NRRL Y-7124 (ARS Culture Collection)

A slant of P. stipitis was prepared from a rehydrated lyophilizedculture obtained from ARS Culture Collection. A portion of the slantmaterial was streaked onto an YM Broth+20 g/L agar (pH 5.0) andincubated at 30° C. for 2 days. A 250 mL Erlenmeyer flask containing 100mL of medium (40 g/L glucose, 1.7 g/L yeast nitrogen base, 2.27 g/Lurea, 6.56 g/L peptone, 40 g/L xylose, pH 5.0) was inoculated with asmall amount of plate material and incubated for 24 hours at 25° C. and125 rpm. After 23 hours of growth, a sample was taken and analyzed foroptical density (600 nm in a UV spectrophotometer) and purity (Gramstain). Based on these results, one flask (called the Seed Flask) at anoptical density of 5.23 and with a clean Gram Stain was chosen toinoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved empty at 121°C. and 15 psi and filter sterilized (0.22 μm filter) media added to theflasks prior to the addition of the test materials. The test materialswere not sterilized, as autoclaving will change the content of thesamples and filter sterilization not appropriate for sterilization ofsolids. The test samples were added at the time of inoculation (ratherthan prior to) to reduce the possibility of contamination. In additionto the test samples, 1 mL (1% v/v) of seed flask material was added toeach flask. The flasks were incubated as described above for 48 hours.

Zymomonas mobilis ATCC 31821 (American Type Culture)

A slant of Z. mobilis was prepared from a rehydrated lyophilized cultureobtained from ATTC. A portion of the slant material was streaked ontoDYE plates (glucose 20 g/L, Yeast Extract 10 g/L, Agar 20 g/L, pH 5.4)and incubated at 30° C. and 5% CO₂ for 2 days. A 20 mL screw-cap testtube containing 15 mL of medium (25 g/L glucose, 10 g/L yeast extract, 1g/L MgSO₄.7H₂O, 1 g/L (NH₄)₂SO₄, 2 g/L KH₂PO₄, pH 5.4) was inoculatedwith one colony and incubated for 24 hours at 30° C. with no shaking.After 23 hours of growth, a sample was taken and analyzed for opticaldensity (600 nm in a UV spectrophotometer) and purity (gram stain).Based on these results, one tube (OD 1.96) was chosen to inoculate thesecond seed flask. The second seed flask was a 125 ml flask containing70 mL of the media described above and was inoculated with 700 μL (1%v/v) and incubated for 24 hours at 30° C. with no shaking. After 23hours of growth, a sample was taken and analyzed for optical density(600 nm in a UV spectrophotometer) and purity (gram stain). Based onthese results, one flask (called the Seed Flask) with an OD of 3.72 waschosen to inoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above with the exception of yeast extract at 5g/L. All flasks were autoclaved empty at 121° C. and 15 psi and filtersterilized (0.22 μm filter) media added to the flasks prior to theaddition of the test materials. The test materials were not sterilized,as autoclaving will change the content of the samples and filtersterilization not appropriate for sterilization of solids. The testsamples were added at the time of inoculation to reduce the possibilityof contamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated asdescribed above for 36 hours

Analysis

Two samples were analyzed for cell concentration (using spread platingfor Z. mobilis and direct counts (haemocytometer and microscope for S.cerevisiae and P. stipitis). Appropriately diluted samples of Z. mobiliswere spread on Dextrose Yeast Extract (glucose 20 g/L, Yeast Extract 10g/L, Agar 20 g/L, pH 5.4) plates, incubated at 30° C. and 5% CO2 for 2days, and the number of colonies counted. Appropriately diluted samplesof S. cerevisiae and P. stipitis were mixed with 0.05% Trypan blue,loaded into a Neubauer haemocytometer. The cells were counted under 40×magnification.

Three samples were analyzed for ethanol concentration using the YSIBiochem Analyzer based on the alcohol dehydrogenase assay (YSI,Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes andthe supernatant stored at −20° C. to preserve integrity. The sampleswere diluted to between 0-3.2 g/L ethanol prior to analysis. A standardof 3.2 g/L ethanol was analyzed approximately every 30 samples to ensurethe integrity of the membrane was maintained during analysis. Theoptical density (600 nm) of the samples is not reported because thesolid test samples interfered with absorbance measurement by increasingthe turbidity of the samples and are inaccurate.

Results of Ethanol Analysis

Performance was used to compare each sample to the control for eachmicroorganism (Tables 37-39). However, the % performance cannot be usedto compare between strains. When comparing strains, the totalconcentration of ethanol should be used. When analyzing the data, a %performance of less than 80% may indicate toxicity when accompanied bylow cell number. The equation used to determine % performance is:% Performance=(ethanol in the sample/ethanol in control)×100

TABLE 37 Ethanol Concentration and % Performance Using Saccharomycescerevisiae 24 hours 30 hours 36 hours Ethanol Ethanol EthanolConcentration % Concentration % Concentration % Sample # (g/L)Performance (g/L) Performance (g/L) Performance P132 4.0 140 5.2 1273.26 176 P132-10 4.2 147 5.1 125 3.86 209 P132-100 4.3 149 5.6 136 3.47187 A132 5.5 191 6.5 160 5.24 283 A132-10 1.9 67 6.3 153 5.54 299A132-100 4.4 154 5.6 137 4.04 218 G132 5.3 186 6.0 146 3.99 215 G132-105.2 180 6.4 156 4.63 250 G132-100 5.5 191 6.3 155 4.60 248 WS132 4.8 1686.3 155 4.51 244 WS132-10 4.9 172 6.0 146 4.55 246 WS132-100 4.9 170 5.7140 4.71 254 Control 2.9 100 4.1 100 1.85 100

TABLE 38 Ethanol Concentration and % Performance Using Pichia stipitis24 hours 36 hours 48 hours Ethanol Ethanol Ethanol Concentration %Concentration % Concentration % Sample # (g/L) Performance (g/L)Performance (g/L) Performance P132 2.8 130 3.4 188 8.1 176 P132-10 7.3344 11.9 655 15.8 342 P132-100 5.2 247 8.6 472 13.3 288 A132 12.2 57514.7 812 14.9 324 A132-10 15.1 710 18.7 1033 26.0 565 A132-100 10.9 51416.7 923 22.2 483 G132 8.0 375 12.9 713 13.3 288 G132-10 10.1 476 16.0884 22.3 485 G132-100 8.6 406 15.2 837 21.6 470 WS132 9.8 460 14.9 82017.9 389 WS132-10 7.8 370 16.1 890 19.3 418 WS132-100 9.1 429 15.0 82915.1 328 Sample A* 13.2 156 19.0 166 20.6 160 Control 2.1 100 1.8 1004.6 100 Samples in BOLD were the highest ethanol producers, over 20 g/Land similar to the concentrations in wood hydrolyzates (H.K. Sreenathand T.W. Jeffries Bioresource Technology 72 (2000) 253-260). *Analyzedin later shake flask experiment.

TABLE 39 Ethanol Concentration and % Performance Using Zymomonas mobilis24 hours 30 hours 36 hours Ethanol Ethanol Ethanol Concentration %Concentration % Concentration % Sample # (g/L) Performance (g/L)Performance (g/L) Performance P132 7.5 85 6.8 84 7.5 93 P132-10 7.5 854.8 59 6.8 84 P132-100 7.3 83 6.2 77 7.1 88 A132 9.6 109 8.3 103 9.1 112A132-10 9.2 105 8.4 105 8.8 109 A132-100 8.2 93 7.6 94 7.6 93 WS132 7.989 7.1 88 7.7 94 WS132-10 8.2 93 6.8 85 7.3 90 WS132-100 8.7 98 6.9 868.3 102 G132 8.7 99 7.1 88 8.1 99 G132-10 7.8 88 7.0 88 7.3 90 G132-1008.6 98 7.8 98 8.3 102 Control 8.8 100 8.0 100 8.1 100Results from Cell Concentration Analysis

% Cells is used to compare each sample to the control for each organism(Tables 40-42). However, the % cells cannot be used to compare betweenstrains. When comparing strains, the total concentration of cells shouldbe used. When analyzing the data, a % performance of less than 70% mayindicate toxicity when accompanied by low ethanol concentration. Theequation used to determine % performance is:% cells=(number of cell in the sample/number of cells in control)×100

TABLE 40 Results from Cell Concentration Analysis for Saccharomycescerevisiae 24 hours 36 hours Cell Concentration % Cell Concentration %Sample # (×10⁸/mL) Cells (×10⁸/mL) Cells P132 1.99 166 2.51 83 P132-102.51 209 1.91 63 P132-100 1.35 113 1.99 66 A132 3.80 316 2.59 85 A132-101.73 144 3.90 129 A132-100 3.98 331 2.51 83 G132 2.14 178 3.12 103G132-10 2.33 194 2.59 85 G132-100 3.57 298 2.66 88 WS132 4.10 341 2.6688 WS132-10 2.63 219 2.81 93 WS132-100 2.29 191 2.40 79 Control 1.20 1003.03 100

TABLE 41 Results from Cell Concentration Analysis for Pichia stipitis 24hours 48 hours Cell Concentration % Cell Concentration % Sample #(×10⁸/mL) Cells (×10⁸/mL) Cells P132 16.4 108 20.3 87 P132-10 11.5 769.5 41 P132-100 6.5 43 17.8 76 A132 7.1 47 10.2 44 A132-10 12.7 84 9.340 A132-100 11.8 78 18.3 78 G132 4.5 30 4.8 21 G132-10 22.8 151 9.8 42G132-100 10.1 67 21.7 93 WS132 17.6 117 8.2 35 WS132-10 5.3 35 10.8 46WS132-100 9.3 62 10.7 46 Control 15.1 100 23.4 100

TABLE 42 Results from Cell Concentration Analysis for Zymomonas mobilis24 hours 36 hours Cell Concentration % Cell Concentration % Sample #(×10⁸/mL) Cells (×10⁸/mL) Cells P132 7.08 86 2.97 66 P132-10 21.80 2644.37 98 P132-100 4.50 54 3.35 75 A132 6.95 84 1.99 44 A132-10 6.13 744.05 91 A132-100 9.60 116 4.20 94 G132 7.48 90 3.84 86 G132-10 14.75 1782.89 65 G132-100 6.00 72 2.55 57 WS132 9.70 117 4.55 102 WS132-10 13.20160 4.32 97 WS132-100 5.15 62 2.89 65 Control 8.27 100 4.47 100

Example 17 Shake Flask Fermentation of Cellulose Samples Using P.stipitis

Summary

Thirteen samples were tested for ethanol production in P. stipitisculture without sugar added. They were tested in the presence andabsence of cellulase (Accellerase 1000® enzyme complex, Genencor).Equipment and reagents used for the experiment are listed below inTables 43-45.

TABLE 43 Equipment and frequency of maintenance Frequency of EquipmentManufacturer Maintenance Shakers (2) B. Braun Biotech, QuarterlyCertomat BS-1 Spectrophotometer Unicam, UV300 Biannual YSI BiochemAnalyzer Interscience, YSI Monthly

TABLE 44 YSI Components used in shake flask study Component Catalog #Lot # YSI Ethanol Membrane 2786 07L100153 YSI Ethanol Standard (3.2 g/L)2790 012711040 YSI Ethanol Buffer 2787 07M1000053, 07100215

TABLE 45 Chemicals used for shake flask fermentation Media ComponentManufacturer Reference # Lot # Urea ScholAR 9472706 AD-7284-43 ChemistryYeast Nitrogen Base Becton Dickinson 291940 7128171 Peptone BectonDickinson 211677 4303198 YM Broth Becton Dickinson 271120 6278265Accellerase ® Genencor Accellerase ® 1600794133 Enzyme complex 1000Xylose BioChemika 95731 1304473 51707231 Glucose Sigma G-5400 107H0245

A slant of P. stipitis NRRL Y-7124 was prepared from a rehydratedlyophilized culture obtained from ARS Culture Collection. A portion ofthe slant material was streaked onto a Yeast Mold (YM) Broth+20 g/L agar(pH 5.0) and incubated at 30° C. for 2 days. A 250 mL Erlenmeyer flaskcontaining 100 mL of medium (40 g/L glucose, 1.7 g/L yeast nitrogenbase, 2.27 g/L urea, 6.56 g/L peptone, 40 g/L xylose, pH 5.0) wasinoculated with one colony and incubated for 24 hours at 25° C. and 100rpm. After 23 hours of growth, a sample was taken and analyzed foroptical density (600 nm in a UV spectrophotometer) and purity (Gramstain). Based on these results, one flask (called the Seed Flask) at anoptical density of 6.79 and with a clean Gram stain was chosen toinoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL ofmedium (1.7 g/L yeast nitrogen base, 2.27 g/L urea, and 6.56 g/Lpeptone). No sugar (glucose or xylose) was added to the growth flaskmedium. All flasks were autoclaved empty at 121° C. and 15 psi andfilter sterilized (0.22 μm filter) media added to the flasks prior tothe addition of the test materials. The test materials were notsterilized, as autoclaving will change the content of the samples andfilter sterilization is not appropriate for sterilization of solids. Thetest samples (listed in Table 46) were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 1 mL (1% v/v) of seed flask material wasadded to each flask. Flasks containing sample P132-100 required theaddition of 0.4 mL 1 M NaOH to bring the pH to 5.0. The flasks wereincubated at 30° C. and 150 rpm above for 96 hours.

One set of duplicate flasks per feedstock contained Accellerase® enzymecomplex (1.25 mL per flask, highest recommended dosage is 0.25 mL pergram of biomass, Genencor) to attempt simultaneous saccharification andfermentation (SSF). The other set of duplicate flasks did not containAccellerase® enzyme complex. A total of 52 flasks were analyzed.

Six control flasks were also analyzed. Positive control flasks containedSolkaFloc 200 NF Powdered Cellulose (lot # UA158072, International FiberCorporation) at a concentration of 2.5 grams per 100 mL flask (25 gramsper L) with and without addition of Accellerase® enzyme complex. Inaddition, a control containing sugars (glucose and xylose) only wasused.

TABLE 46 The amount of each feedstock added to each flask Amount addedto Flask Xyleco Number (g/100 mL) P132 2.5 P132-10 2.5 P132-100 2.5 A1325 A132-10 5 A132-100 5 G132 5 G132-10 5 G132-100 5 WS132 5 WS132-10 5WS132-100 5 Sample A 5Analysis

Samples were analyzed for ethanol concentration (Tables 47, 48, and 49)using the YSI Biochem Analyzer based on the alcohol dehydrogenase assay(YSI, Interscience). Samples were centrifuged at 14,000 rpm for 20minutes and the supernatant stored at −20° C. The samples were dilutedto between 0-3.2 g/L ethanol prior to analysis. A standard of 2.0 g/Lethanol was analyzed approximately every 30 samples to ensure theintegrity of the membrane was maintained during analysis.

Results

TABLE 47 Results of Control Flasks Ethanol Concentration (g/L) Control24 hours 36 hours 48 hours 96 hours Containing Glucose, no 13.20 19.0020.60 21.60 cellulose, no enzyme Containing Crystalline 0.00 0.00 0.000.00 Cellulose (Solka Floc), no sugar, no enzyme Containing Crystalline6.56 7.88 9.80 8.65 Cellulose (Solka Floc) at 25 g/L, no sugar,Accellerase ® added

TABLE 48 Results of Shake Flasks without Accellerase ® 1000 EnzymeComplex Sample Ethanol Concentration (g/L) Number 24 hours 36 hours 48hours 96 hours P132 0.09 0.00 0.00 0.12 P132-10 0.02 0.01 0.02 0.17P132-100 0.09 0.01 0.00 0.02 A132 1.74 1.94 2.59 3.70 A132-10 1.82 2.362.30 2.96 A132-100 0.30 0.73 1.31 2.38 G132 0.40 0.09 0.24 0.42 G132-100.69 0.42 0.22 0.24 G132-100 0.19 0.05 0.05 0.21 WS132 0.47 0.50 0.680.65 WS132-10 0.47 0.49 0.34 0.92 WS132-100 0.14 0.07 0.08 0.22 Sample A1.88 1.89 2.30 3.28

TABLE 49 Results of Shake Flasks with Accellerase ® 1000 Enzyme ComplexSample Ethanol Concentration (g/L) Number 24 hours 36 hours 48 hours 96hours P132 7.04 8.72 9.30 5.80 P132-10 4.22 4.48 4.49 1.24 P132-100 3.184.28 4.70 3.35 A132 2.79 2.91 2.03 4.30 A132-10 3.31 1.62 2.11 2.71A132-100 2.06 1.92 1.02 1.47 G132 0.87 0.40 0.32 0.44 G132-10 1.38 1.040.63 0.07 G132-100 2.21 2.56 2.34 0.12 WS132 1.59 1.47 1.07 0.99WS132-10 1.92 1.18 0.73 0.23 WS132-100 2.90 3.69 3.39 0.27 Sample A 2.212.35 3.39 2.98

Example 18 Cellulase Assay

Summary

Thirteen samples were tested for cellulase susceptibility using anindustry cellulase (Accellerase® 1000, Genencor) under optimumconditions of temperature and pH.

Protocol

The protocol is a modification of the NREL “Laboratory AnalyticalProcedure LAP-009 Enzymatic Saccharification of LignocellulosicBiomass”. A sample of material was added to 10 mL 0.1 M sodium citratebuffer (pH 4.8) and 40 mg/mL tetracycline (to prevent growth ofbacteria) in a 50 mL tube in duplicate. The amount of sample added toeach tube is listed in Table 50. Some samples were difficult to mix(P132, P132-10, P132-100), so were added at a lower concentration. Apositive control of 0.2 grams SolkaFloc 200 NF Powdered Cellulose (lot #UA158072, International Fiber Corporation) and a negative control (nosample) were also included. Enough reverse osmosis (RO) water to bringthe volume to a total of 20 mL was added to the tubes. Both the sodiumcitrate buffer and water were heated to 50° C. prior to use.

Accellerase® 1000 enzyme was added to each tube at a dosage of 0.25 mLper gram of biomass (highest dosage recommended by Genencor). The tubeswere incubated at 45° angle at 150 rpm and 50 degrees C. (recommended byGenencor) for 72 hours. Samples were taken at 0, 3, 6, 9, 12, 18, 24,48, and 72 hours (Table 52 and 53), centrifuged at 14,000 rpm for 20minutes and the supernatant frozen at −20° C. The glucose concentrationin the samples was analyzed using the YSI Biochem Analyzer(Interscience) using the conditions described in Table 51. A glucosestandard solution of 2.5 g/L was prepared by dissolving 2.500 gramsglucose (Sigma Cat# G7528-5KG, Lot#:107H0245) in distilled water. Oncedissolved, the total volume was brought to 1 L with distilled water in avolumetric flask. The standard was prepared fresh weekly and stored at4° C.

TABLE 50 Amount of Each Sample Added Xyleco Number Amount added to Tube(g/20 mL) P132 0.5 P132-10 0.5 P132-100 0.5 A132 0.75 A132-10 0.75A132-100 0.75 G132 0.75 G132-10 0.75 G132-100 0.75 WS132 0.75 WS132-100.75 WS132-100 0.75 Sample A 0.75 SolkaFloc 200NF (Control) 0.2 NegativeControl 0

TABLE 51 YSI Components Used in Shake Flask Study Component Catalog #Lot # YSI Glucose Membrane 2365 07D100124 YSI Glucose Buffer 2357014614AResults

TABLE 52 Cellulase Assay Results Sample Glucose Concentration (mg/mL) atIncubation Time (hours) Number 0 3 6 9 12 18 24 48 72 P132 0.59 4.197.00 8.72 9.70 10.95 12.19 15.10 15.65 P132-10 0.36 3.37 5.08 6.39 6.987.51 8.99 11.25 11.65 P132-100 0.91 3.86 5.67 7.31 8.08 9.47 10.70 12.7013.80 A132 0.39 1.51 1.92 2.40 2.64 3.04 3.30 3.90 4.06 A132-10 0.421.80 2.27 2.63 2.86 3.16 3.43 4.02 4.14 A132-100 0.46 2.09 2.72 3.163.43 3.78 4.09 4.84 5.26 G132 0.40 1.16 1.35 1.52 1.60 1.67 1.85 2.102.21 G132-10 0.34 1.34 1.64 1.95 2.03 2.09 2.36 2.77 3.02 G132-100 0.611.84 2.32 2.89 3.14 3.52 3.97 4.81 5.44 WS132 0.35 1.48 1.81 2.14 2.262.50 2.70 3.18 3.26 WS132-10 0.44 1.77 2.22 2.60 2.76 2.61 3.15 3.623.82 WS132-100 0.70 2.76 3.63 4.59 4.78 5.29 5.96 6.99 7.43 Sample A0.42 1.09 1.34 1.55 1.69 1.66 2.17 2.96 3.71 Negative 0.03 0.03 0.010.01 0.02 0.01 0.02 0.02 0.02 Control (no sample) Positive 0.17 2.383.65 4.71 5.25 5.98 7.19 9.26 9.86 Control (SolkaFloc)

The amount of cellulose digested in the tube was calculated as follows:g/mL glucose×20 mL (volume of sample)×0.9 (to correct for the watermolecule added upon hydrolysis of cellulose)

The percent of the total sample released as glucose (in Table 53 below)was calculated as follows:g of cellulose digested/g of sample added (see Table 5 for details)*100

TABLE 53 Cellulase Assay Results Sample Percent of the Total SampleReleased as Glucose (%) at Incubation Time (h) Number 0 3 6 9 12 18 2448 72 P132 2.02 14.98 25.16 31.36 34.85 39.38 43.81 54.29 56.27 P132-101.19 12.02 18.25 22.97 25.06 27.00 32.29 40.43 41.87 P132-100 3.17 13.7920.38 26.28 29.02 34.06 38.45 45.65 49.61 A132 0.86 3.55 4.58 5.74 6.297.27 7.87 9.31 9.70 A132-10 0.94 4.25 5.42 6.29 6.82 7.56 8.18 9.60 9.89A132-100 1.03 4.94 6.50 7.56 8.18 9.05 9.77 11.57 12.58 G132 0.89 2.713.22 3.62 3.79 3.98 4.39 4.99 5.26 G132-10 0.74 3.14 3.91 4.66 4.82 4.995.62 6.60 7.20 G132-100 1.39 4.34 5.54 6.91 7.49 8.42 9.48 11.50 13.01WS132 0.77 3.48 4.32 5.11 5.38 5.98 6.43 7.58 7.78 WS132-10 0.98 4.185.30 6.22 6.58 6.24 7.51 8.64 9.12 WS132-100 1.61 6.55 8.69 10.99 11.4212.67 14.26 16.73 17.78 Sample A 0.94 2.54 3.19 3.70 4.01 3.96 5.16 7.068.86 Positive 1.29 21.15 32.72 42.30 47.07 53.73 64.53 83.16 88.56Control (SolkaFloc)

Example 19 Shake Flask Fermentation Using Pichia stipitis

Summary

Shake flask fermentation using Pichia stipitis was performed using fourcellulosic materials having the highest % performance from Table 36.

Protocol

Experiments were run under the parameters outlined in Tables 54-56.

TABLE 54 Equipment and Frequency of Maintenance Frequency of EquipmentManufacturer, Name Maintenance Shakers (2) B. Braun Biotech, QuarterlyCertomat BS-1 Spectrophotometer Unicam, UV300 Biannual YSI BiochemAnalyzer Interscience, YSI Monthly

TABLE 55 YSI Components Used in Shake Flask Study Component Reference #Lot # YSI Ethanol Membrane 2786 07M100361 YSI Ethanol Standard (3.2 g/L)2790 1271040 YSI Ethanol Buffer 2787 07J100215

TABLE 56 Chemicals Used for Shake Flask Fermentation Media ComponentManufacturer Reference # Lot # Urea ScholAR Chemistry 9472706 AD-7284-43Yeast Nitrogen Base Becton Dickinson 291940 7128171 Peptone BectonDickinson 211677 4303198 YM Broth Becton Dickinson 271120 6278265 XyloseAlfa Aesar A10643 10130919 Glucose Fisher Scientific BP350-1 030064Seed Development

For all the following shake flask experiments the seed flasks wereprepared using the following procedure.

A working cell bank of P. stipitis NRRL Y-7124 was prepared from arehydrated lyophilized culture obtained from ARS Culture Collection.Cryovials containing P. stipitis culture in 15% v/v glycerol were storedat −75° C. A portion of the thawed working cell bank material wasstreaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH 5.0) and incubatedat 30° C. for 2 days. The plates were held for 2 days at 4° C. beforeuse. A 250 mL Erlenmeyer flask containing 100 mL of medium (40 g/Lglucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone,40 g/L xylose, pH 5.0) was inoculated with one colony and incubated for24 hours at 25° C. and 100 rpm. After 23 hours of growth, a sample wastaken and analyzed for optical density (600 nm in a UVspectrophotometer) and purity (Gram stain). Based on these results, oneflask (called the Seed Flask) at an optical density of between 4 and 8and with a clean Gram stain was used to inoculate all of the testflasks.

Three experiments were run using samples A132-10, A132-100, G132-10, andG132-100. Experiment #1 tested these four samples for ethanolconcentration at varying concentrations of xylose and at constantconcentrations of glucose. Experiment #2 tested these four samples forethanol concentration at double the concentration of feedstock used inthe experiments of Table 36. Finally, experiment #3 tested these foursamples for ethanol concentration while varying both the xylose and theglucose concentrations, simultaneously.

Experiment #1—Varying the Xylose Concentration

Four cellulosic samples (A132-10, A132-100, G132-10, and G132-100) weretested at varying xylose concentrations as listed in Table 57 below.

TABLE 57 Media Composition of Experiment #1 Flasks Xylose ConcentrationGlucose Concentration Treatment (g/L) (g/L) 100% Xylose  40.0 40.0 50%Xylose 20.0 40.0 25% Xylose 10.0 40.0 10% Xylose 4.0 40.0  0% Xylose 0.040.0

The test vessels (a total of 40, 250 mL Erlenmeyer flasks) contained 100mL of medium. Five different types of media were prepared with theamount of xylose and glucose outlined in Table 57. In addition, themedia contained 1.7 g/L yeast nitrogen base (Becton Dickinson #291940)2.27 g/L urea (ScholAR Chemistry #9472706), and 6.56 g/L peptone (BectonDickinson #211677). All flasks were autoclaved empty at 121° C. and 15psi and filter sterilized (0.22 μm filter) media was added to the flasksprior to the addition of the test materials. Flasks were held at roomtemperature for 4 days and inspected for contamination (cloudiness)prior to use. The test materials were not sterilized, as autoclavingwill change the content of the samples and filter sterilization notappropriate for sterilization of solids. The test samples (A132-10,A132-100, G132-10, and G132-100 at 5 g per 100 mL) were added at thetime of inoculation (rather than prior to) to reduce the possibility ofcontamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated at 30°C. and 150 rpm for 72 hours.

Unfortunately, one flask (sample A132-100 with 100% Xylose) was brokenduring the testing. Therefore, all results past 24 hours of incubationare reported as a single flask. After 72 hours of incubation, 100% ofthe original amount of cellulosic material (5.0 g) was added to the 100%Xylose flasks (7 flasks in total, one flask containing sample A132-100was broken) and incubated as above for an additional 48 hours.

TABLE 58 Addition of Feedstock to 100% Xylose Flasks at Incubation Time72 hours Feedstock Added at 72 hours (grams) A132-10 5 A132-100 5G132-10 5 G132-100 5Analysis

Samples were taken from the 40 test flasks at incubation times of 0, 6,12, 24, 36, 48, and 72 hours. In addition, samples were taken at 24 and48 hours post-addition of the second feedstock amount in the 100% Xyloseflasks (see Table 58).

A total of 292 samples were analyzed for ethanol concentration using aYSI Biochem Analyzer based on the alcohol dehydrogenase assay (YSI,Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes andthe supernatant stored at −20° C. Of note, time 0 samples requiredfiltration through a 0.45 μm syringe filter. The samples will be dilutedto between 0-3.2 g/L ethanol prior to analysis. A standard of 2.0 g/Lethanol was analyzed approximately every 30 samples to ensure theintegrity of the membrane was maintained.

A total of 47 samples were analyzed for cell count. Samples will betaken at 72 hours incubation and 48 hours post-addition of morecellulosic material. Appropriately diluted samples were mixed with 0.05%Trypan blue and loaded into a Neubauer haemocytometer. The cells werecounted under 40× magnification.

Experiment #2—Analysis of 2× Feedstock Concentration

The test vessels (a total of 8, 250 mL Erlenmeyer flasks) contained 100mL of medium. The media contained 40 g/L glucose, 40 g/L xylose, 1.7 g/Lyeast nitrogen base (Becton Dickinson #291940) 2.27 g/L urea (ScholARChemistry #9472706), and 6.56 g/L peptone (Becton Dickinson #211677).Flasks were prepared as in Experiment #1. The test samples (A132-10,A132-100, G132-10, and G132-100 at 10 g per 100 mL) were added at thetime of inoculation (rather than prior to) to reduce the possibility ofcontamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated at 30°C. and 150 rpm above for 72 hours.

Analysis

Samples were from the 8 test flasks at an incubation time of 0, 6, 12,24, 36, 48, and 72 hours. Ethanol analyses of the 56 samples wereperformed as per experiment #1 and are reported in Table 59. A cellcount was performed on the 72 hour sample as per experiment #1 and ispresented in Table 60.

TABLE 59 Ethanol Concentration in Flasks with Double Feedstock SampleEthanol Concentration (g/L) Time A132-10 A132-100 G132-10 G132-100 01.38 0.26 0.12 0.11 6 1.75 0.21 0.20 0.10 12 2.16 0.73 0.69 0.31 2419.05 15.35 16.55 12.60 36 21.75 17.55 18.00 15.30 48 26.35 23.95 24.6520.65 72 26.95 27.35 28.90 27.40

TABLE 60 Cell Concentration at 72 hour Incubation Time in Flasks withDouble Feedstock Sample Cell Concentration (×10⁸/mL) A132-10 4.06A132-100 5.37 G132-10 5.18 G132-100 4.47Experiment #3—Varying Xylose and Glucose Concentrations

Four cellulosic samples (A132-10, A132-100, G132-10, and G132-100) weretested at varying xylose and glucose concentrations as listed in thetable below (Table 60).

TABLE 61 Media Composition of Experiment #3 Flasks Xylose ConcentrationGlucose Concentration Treatment (g/L) (g/L) 50% Sugar 20.0 20.0 25%Sugar 10.0 10.0 10% Sugar 4.0 4.0  0% Sugar 0.0 0

The test vessels (a total of 32, 250 mL Erlenmeyer flasks) contained 100mL of medium. Four different types of media were prepared with theamount of xylose and glucose outlined in Table 61. In addition, themedia contained 1.7 g/L yeast nitrogen base (Becton Dickinson #291940)2.27 g/L urea (ScholAR Chemistry #9472706), and 6.56 g/L peptone (BectonDickinson #211677). The flasks were prepared as per Experiment #1. Thetest samples (A132-10, A132-100, G132-10, and G132-100) were added atthe time of inoculation (rather than prior to) to reduce the possibilityof contamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated at 30°C. and 150 rpm for 72 hours.

Analysis

Samples were taken from the 32 test flasks at an incubation time of 0,6, 12, 24, 36, 48, and 72 hours (see Tables 62-65). A total of 224samples were analyzed for ethanol concentration using the YSI BiochemAnalyzer based on the alcohol dehydrogenase assay (YSI, Interscience).Samples were centrifuged at 14,000 rpm for 20 minutes and thesupernatant stored at −20° C. Of note, some of the samples requiredcentrifugation and then filtration through a 0.45 μm syringe filter. Thesamples were diluted to between 0-3.2 g/L ethanol prior to analysis. Astandard of 2.0 g/L ethanol was analyzed approximately every 30 samplesto ensure the integrity of the YSI membrane was maintained.

TABLE 62 Ethanol Results Sample A132-10 Ethanol Concentration (g/L)Sample 0% 10% 25% 50% 100% 0% 10% 25% 50% Time Xylose Xylose XyloseXylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.43 0.42 0.42 0.41 0.390.53 0.57 0.56 0.56 6 1.16 1.16 1.15 1.16 1.12 0.93 0.91 0.83 0.88 121.72 1.86 1.71 1.79 1.90 1.21 2.13 2.47 2.32 24 15.55 15.90 17.05 17.0516.95 1.02 4.88 9.77 13.35 36 17.10 17.40 20.25 21.35 20.25 1.29 4.279.99 17.55 48 16.40 17.05 19.70 23.00 26.80 1.47 3.03 8.33 16.60 7215.15 15.55 19.25 21.85 28.00 1.14 1.52 5.08 14.20 24 — — — — 23.15 — —— — hours post- addition 48 — — — — 21.55 — — — — hours post- addition*Analysis from experiment #3.

TABLE 63 Ethanol Results Sample A132-100 Ethanol Concentration (g/L)Sample 0% 10% 25% 50% 100% 0% 10% 25% 50% Time Xylose Xylose XyloseXylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.11 0.09 0.17 0.20 0.180.12 0.14 0.09 0.13 6 0.13 0.15 0.15 0.15 0.14 0.10 0.11 0.11 0.13 120.88 1.00 1.18 1.25 0.89 0.18 1.58 1.55 1.57 24 15.90 15.70 16.50 16.0514.60** 0.18 3.33 7.99 11.15 36 16.00 17.90 16.90 19.45 17.80** 0.212.85 8.37 16.10 48 15.75 16.70 19.30 22.15 27.00** 0.54 1.47 7.54 15.6072 14.85 15.35 18.55 21.30 28.50** 0.78 0.51 4.47 12.90 24 — — — —24.80** — — — — hours post- addition 48 — — — — 23.60** — — — — hourspost- addition *Analysis from experiment #3. **All results based onanalysis of one flask.

TABLE 64 Ethanol Results Sample G132-10 Ethanol Concentration (g/L)Sample 0% 10% 25% 50% 100% 0% 10% 25% 50% Time Xylose Xylose XyloseXylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.09 0.08 0.08 0.08 0.080.05 0.05 0.05 0.06 6 0.14 0.13 0.14 0.14 0.13 0.11 0.12 0.11 0.12 121.01 0.96 1.00 0.87 1.14 0.48 1.60 1.79 1.71 24 15.90 15.70 16.30 16.0514.60 0.13 3.96 8.54 11.10 36 15.10 17.45 16.80 18.75 22.15 0.09 3.028.69 16.55 48 15.95 16.90 19.25 21.10 24.00 0.07 2.05 8.10 16.50 7213.50 15.80 18.55 21.25 26.55 0.09 0.11 5.55 14.15 24 — — — — 24.95 — —— — hours post- addition 48 — — — — 24.20 — — — — hours post- addition*Analysis from experiment #3.

TABLE 65 Ethanol Results Sample G132-100 Ethanol Concentration (g/L) 10%25% 50% Sample 0% w/v w/v w/v 100% w/v 0% 10% 25% 50% Time Xylose XyloseXylose Xylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.04 0.04 0.040.04 0.05 0.05 0.05 0.05 0.06 6 0.07 0.07 0.08 0.08 0.07 0.04 0.05 0.050.06 12 0.60 0.56 0.67 0.58 0.71 0.13 1.37 1.48 1.44 24 13.05 14.4514.90 13.95 12.05 0.03 3.67 7.62 10.55 36 15.10 17.10 18.25 18.20 19.250.01 3.09 8.73 16.10 48 14.40 17.00 19.35 22.55 24.45 0.01 1.91 7.7615.85 72 14.70 15.40 18.45 22.10 27.55 0.03 0.01 5.08 14.30 24 hours — —— — 25.20 — — — — post- addition 48 hours — — — — 24.60 — — — — post-addition *Analysis from experiment #3.

Samples were taken at 72 hours incubation for cell counts (see Tables66-67). Appropriately diluted samples were mixed with 0.05% Trypan blueand loaded into a Neubauer haemocytometer. The cells were counted under40× magnification.

Results

One seed flask was used to inoculate all Experiment #1 and #2 testflasks. The optical density (600 nm) of the seed flask was measured tobe 5.14 and the cell concentration was 4.65×10⁸ cells/mL (Tables 65-66).Therefore, the initial concentration of cells in the test flasks wasapproximately 4.65×10⁶ cells/mL.

A second seed flask was used to inoculate Experiment #3 flasks. Theoptical density (600 nm) of the seed flask was 5.78 and the cellconcentration was 3.75×10⁸ cells/mL. Therefore, the initialconcentration of cells in the test flasks was approximately 3.75×10⁶cells/mL.

TABLE 66 Cell Counts at Incubation Time of 72 hours Cell Concentration(×10 ⁸ /mL) 0% 10% 25% 50% 100% 0% 10% 25% 50% Sample Xylose XyloseXylose Xylose Xylose Sugar Sugar Sugar Sugar A132-10 0.37 0.63 3.72 4.924.05 0.26 0.22 0.26 1.54 A132-100 0.99 1.07 0.99 0.78 1.97 0.03* 0.330.44 1.81 G132-10 0.95 4.50 2.67 2.67 3.82 0.01* 0.17 0.49 1.92 G132-1006.53 4.02 4.84 4.47 5.29 0.01* 0.33 0.89 2.22 *Samples were heavilycontaminated after 72 hours of growth. This is expected because thePichia did not grow well without sugar added, and contaminants (from thenon-sterile samples) were able to out-grow the Pichia.

TABLE 67 Cell Counts at Incubation Time of 48 hours Post-Addition (100%Xylose and Glucose) Sample Cell Concentration (×10⁸/mL) A132-10 10.17A132-100 3.38 G132-10 3.94 G132-100 6.53

Example 20 Toxicity Testing of Lignocellulosic Samples Against P.stipitis and S. cerevisiae

Summary

Thirty-seven samples were analyzed for toxicity against twoethanol-producing cultures, Saccharomyces cerevesiae and Pichiastipitis. In this study, glucose was added to the samples in order todistinguish between starvation of the cultures and toxicity of thesamples.

TABLE 68 Conditions for Toxicity Testing Organism Saccharomyces Pichiacerevisiae stipitis Variable ATCC 24858 NRRL Y-7124 Inoculation Volume(mL) 0.5-1 (target 6-7 × 1 (target 3-4 × 10⁵ cells/mL) 10⁶ cells/mL)Test Repetition Single Flasks Incubation Temperature 25° C. 25° C. (±1°C.) Shaker Speed (rpm) 200 125 Type of Container 500 mL 250 mLErlenmeyer Erlenmeyer Flask Flask Media volume 100 mL 100 mL TotalIncubation time  72  72 (hours) Ethanol Analysis 0, 6, 12, 24, 36, 48,72 0, 6, 12, 24, 36, 48, 72 (hours) Cell Counts (hours) 24, 72 24, 72 pH0 hours 0 hoursProtocol

A summary of the protocol used is listed in Table 68. A description ofthe chemicals used in toxicity testing is listed in Table 69. Twocontrol flasks (no sample added) were performed for each microorganismfor each week of testing. A total of 82 flasks were analyzed.

During the experiments, no ethanol or cells appeared in the P. stipitisflasks containing samples C, C-1e, C-5e, and C-10e in the first 24 hoursof incubation. In order to confirm the results, the test was repeated.The second test confirmed some inhibition of P. stipitis growth whensamples C, C1E, C5E, and C10E were added to the flasks.

TABLE 69 Chemicals and Materials Used for Toxicity Testing MediaComponent Manufacturer Reference # Lot # Urea ScholAR Chemistry 9472706AD-7284-43 Yeast Nitrogen Base Becton Dickinson 291940 7128171 PeptoneBecton Dickinson 211677 4303198 Xylose Alfa Aesar A10643 10130919Glucose Sigma G-5400 107H0245 Yeast Extract Becton Dickinson 2886204026828 YM Broth Becton Dickinson 271120 6278265

TABLE 70 YSI Components Used in Toxicity Study Component Catalogue # YSIEthanol Membrane 2786 YSI Ethanol Standard (3.2 g/L) 2790 YSI EthanolBuffer 2787Test Samples

Seven test samples (all with the C designation) were ground using acoffee grinder suitable for small samples. The samples were ground to aconsistent particle size (between samples) with the naked eye. Samplenumber C-100e ground easily to a small particle size.

All samples were added to the flasks at a concentration of 50 grams perliter with the exception of the six P samples (25 grams per liter).These samples were white to off-white in color and visually fluffy andthe flasks would not mix properly (not enough free liquid) at the 50grams per liter concentration. Samples S dissolved easily and could inthe future be added to the flasks at a higher concentration. Samples Aand G could be added at 100 grams per liter in the future.

Testing was performed using the two microorganisms as described below.

Saccharomyces cerevisiae ATCC 24858 (American Type Culture Collection)

A working cell bank of S. cerevisiae ATCC 24858 was prepared from arehydrated lyophilized culture obtained from American Type CultureCollection. Cryovials containing S. cerevisiae culture in 15% v/vglycerol are stored at −75° C. A portion of the thawed working cell bankmaterial will be streaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH5.0) and incubated at 30° C. for 2 days. A 250 mL Erlenmeyer flaskcontaining 50 mL of medium (20 g/L glucose, 3 g/L yeast extract, and 5.0g/L peptone, pH 5.0) was inoculated with one colony from the YM plateand incubated for 24 hours at 25° C. and 200 rpm. After 23 hours ofgrowth, a sample was taken and analyzed for optical density (600 nm in aUV spectrophotometer) and purity (Gram stain). Based on these results,one flask (called the Seed Flask) with an OD of 9-15 and pure Gram stainwas to be used for inoculating the growth flasks. After 23 hours ofgrowth, the seed flask had a low OD (5.14) and cell count (1.35×10⁸cells/mL). Of note, the colony taken from the seed plate was smallerthan usual. Therefore, 0.5 mL of seed material (as opposed to theplanned 0.1 mL) was added to each test vessel.

The test vessels were 500 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved at 121° C.and 15 psi prior to the addition of the test materials. The testmaterials were not sterilized, as autoclaving would change the contentof the samples. The test samples were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 0.5-1.0 mL (0.5-1.0% v/v) of seed flaskmaterial was added to each flask. The flasks were incubated as describedabove for 72 hours.

Pichia stipitis (ARS Culture Collection)

A working cell bank of P. stipitis NRRL Y-7124 was prepared from arehydrated lyophilized culture obtained from ARS Culture Collection.Cryovials containing P. stipitis culture in 15% v/v glycerol are storedat −75° C. A portion of the thawed working cell bank material wasstreaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH 5.0) and incubatedat 30° C. for 2 days. The plates were held for up to 5 days at 4° C.before use. A 250 mL Erlenmeyer flask containing 100 mL of medium (40g/L glucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/Lpeptone, 40 g/L xylose, pH 5.0) was inoculated with one colony andincubated for 24 hours at 25° C. and 125 rpm. After 23 hours of growth,a sample was taken and analyzed for optical density (600 nm in a UVspectrophotometer) and purity (Gram stain). Based on these results, oneflask (called the Seed Flask) at an optical density of 5-9 and with apure Gram Stain was used to inoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved empty at 121°C. and 15 psi and filter sterilized (0.22 μm filter) medium added to theflasks prior to the addition of the test materials. The test materialswere not sterilized, as autoclaving would change the content of thesamples and filter sterilization not appropriate for sterilization ofsolids. The test samples were added at the time of inoculation (ratherthan prior to) to reduce the possibility of contamination. In additionto the test samples, 1 mL (1% v/v) of seed flask material was added toeach flask. The flasks were incubated as described above for 72 hours.

Analysis

Samples were taken from seed flasks just prior to inoculation and eachtest flask at 24 and 72 hours and analyzed for cell concentration usingdirect counts. Appropriately diluted samples of S. cerevisiae and P.stipitis were mixed with 0.05% Trypan blue, loaded into a Neubauerhaemocytometer. The cells were counted under 40× magnification.

Samples were taken from each flask at 0, 6, 12, 24, 36, 48 and 72 hoursand analyzed for ethanol concentration using the YSI Biochem Analyzerbased on the alcohol dehydrogenase assay (YSI, Interscience). Sampleswere centrifuged at 14,000 rpm for 20 minutes and the supernatant storedat −20° C. The samples will be diluted to 0-3.2 g/L ethanol prior toanalysis. A standard of 2.0 g/L ethanol was analyzed approximately every30 samples to ensure the integrity of the membrane was maintained duringanalysis.

Calculations

The following calculations were used to compare the cell counts andethanol concentration to the control flasks.% performance=(concentration of ethanol in test flask/ethanol incontrol)*100%cells=(number of cells in test flask/number of cells in controlflask)*100Results

The S. cerevisiae seed flask had an optical density (600 nm) of 5.14 anda cell concentration of 1.35×10⁸ cells/mL. One half mL of seed flaskmaterial was added to each of the test flasks. Therefore, the startingcell concentration in each flask was 6.75×10⁵/mL. During the second weekof testing, the S. cerevisiae seed flask had an optical density (600 nm)of 4.87 and a cell concentration of 3.15×10⁷ cells/mL. One mL of seedflask material was added to each of the test flasks. Therefore, thestarting cell concentration in each flask was 6.30×10⁵/mL. The pH of theS. cerevisiae flasks at a sample time of 0 hours is presented in Table71. The pH of the flask contents was within the optimal pH for S.cerevisiae growth (pH 4-6). No pH adjustment was required.

TABLE 71 pH of S. cerevisiae flasks at sample time 0 hours Sample NumberpH P 5.04 P1E 4.99 P5E 5.04 P10E 4.98 P50E 4.67 P100E 4.43 G 5.45 G1E5.47 G5E 5.46 G10E 5.39 G50E 5.07 A 5.72 A1E 5.69 A5E 5.62 A10E 5.61A50E 5.74 S* 5.10 S1E 5.08 S5E 5.07 S10E 5.04 S30E 4.84 S50E 4.57 S100E4.33 C 5.46 C1E 5.54 C5E 5.50 C10E 5.33 C30E 5.12 C50E 4.90 C100E 4.66ST 5.11 ST1E 5.06 ST5E 4.96 ST10E 4.94 ST30E 5.68 ST50E 4.48 ST100E 4.23control A 5.02 control B 5.04 *“S” refers to sucrose *“C” refers to corn*“ST” refers to starch

The ethanol concentration and performance in the S. cerevisiae flasksare presented in Table 72 and 73. The highest ethanol concentrationswere produced by the S series.

TABLE 72 Ethanol Concentration in S. cerevisiae flasks Sample EthanolConcentration (g/L) at the following times (hours) Number 0 6 12 24 3648 72 P 0.02 0.04 0.38 5.87 7.86 5.41 1.04 P1E 0.03 0.03 0.28 5.10 8.035.46 0.58 P5E 0.03 0.04 0.57 8.84 6.38 3.40 0.04 P10E 0.06 0.05 0.656.63 7.66 5.57 1.40 P50E 0.04 0.03 0.26 2.80 5.85 8.59 5.68 P100E 0.040.02 0.12 3.64 8.26 7.51 3.03 G 0.04 0.04 0.57 10.20 8.24 6.66 2.84 G1E0.04 0.05 0.46 10.20 9.24 6.94 2.84 G5E 0.11 0.11 0.44 10.00 8.7 6.360.88 G10E 0.05 0.04 0.40 9.97 8.41 5.79 0.11 G50E 0.05 0.05 0.48 9.728.33 6.13 2.38 A 0.29 0.38 0.48 8.43 8.76 7.09 4.66 A1E 0.34 0.44 0.799.66 8.9 7.18 2.64 A5E 0.55 0.45 0.99 9.44 8.96 7.56 3.80 A10E 0.55 0.550.93 9.58 8.33 6.28 1.40 A50E 0.22 0.08 0.38 9.38 8.01 5.99 0.98 S 0.030.03 0.39 5.73 7.06 10.10 15.90 S1E 0.05 0.06 0.31 7.24 9.52 12.10 14.90S5E 0.02 0.05 0.34 5.87 7.68 11.90 19.00 S10E 0.03 0.04 0.35 5.88 7.7211.50 19.30 S30E 0.03 0.05 0.09 5.94 7.97 11.20 20.40 S50E* 0.13 0.190.47 5.46 7.96 13.00 18.30 S100E 0.11 0.10 0.21 7.00 10.6 13.80 12.70 C0.01 0.04 0.32 8.47 7.57 5.48 6.40 C1E 0.00 0.06 0.37 8.93 7.86 5.991.37 C5E 0.03 0.05 0.48 9.32 7.92 5.69 1.41 C10E 0.02 0.04 0.52 9.147.67 5.34 0.35 C30E 0.02 0.05 0.28 9.15 8.15 5.84 2.47 C50E 0.03 0.060.44 9.31 7.79 5.78 1.79 C100E 0.03 0.06 0.58 9.06 6.85 5.95 1.09 ST0.02 0.05 0.99 8.54 6.69 5.09 0.42 ST1E 0.03 0.04 0.70 8.87 7.29 4.811.04 ST5E 0.02 0.04 0.52 8.61 7.16 4.97 0.85 ST10E 0.02 0.05 0.33 8.977.05 5.26 0.68 ST30E 0.03 0.04 0.71 8.47 6.96 4.89 0.21 ST50E 0.04 0.070.34 8.46 8.19 7.04 3.20 ST100E 0.03 0.10 0.30 9.30 8.62 7.29 4.23control A 0.01 0.07 0.85 5.92 8.18 7.81 6.26 control B 0.01 0.04 0.274.86 6.43 8.01 6.75 control A* 0.04 0.21 1.36 5.19 7.31 7.55 5.16control B* 0.03 0.20 1.18 5.16 5.96 7.62 5.32 *analyzed week 2See Table 72 for Sample Number key

TABLE 73 Performance in S. cerevisiae flasks Sample Performance (%) atthe following times (hours) Number 24 36 48 72 P 108.9 107.6 68.4 16.0P1E 94.6 109.9 69.0 8.9 P5E 164.0 87.3 43.0 0.6 P10E 123.0 104.9 70.421.5 P50E 51.9 80.1 108.6 87.3 P100E 67.5 113.1 94.9 46.5 G 189.2 112.884.2 43.6 G1E 189.2 126.5 87.7 43.6 G5E 185.5 119.1 80.4 13.5 G10E 185.0115.1 73.2 1.7 G50E 180.3 114.0 77.5 36.6 A 156.4 119.9 89.6 71.6 A1E179.2 121.8 90.8 40.6 A5E 175.1 122.7 95.6 58.4 A10E 177.7 114.0 79.421.5 A50E 174.0 109.7 75.7 15.1 S 106.3 96.6 127.7 244.2 S1E 134.3 130.3153.0 228.9 S5E 108.9 105.1 150.4 291.9 S10E 109.1 105.7 145.4 296.5S30E 110.2 109.1 141.6 313.4 S50E* 105.5 119.9 171.3 349.2 S100E 129.9145.1 174.5 195.1 C 157.1 103.6 69.3 98.3 C1E 165.7 107.6 75.7 21.0 C5E172.9 108.4 71.9 21.7 C10E 169.6 105.0 67.5 5.4 C30E 169.8 111.6 73.837.9 C50E 172.7 106.6 73.1 27.5 C100E 168.1 93.8 75.2 16.7 ST 158.4 91.664.3 6.5 ST1E 164.6 99.8 60.8 16.0 ST5E 159.7 98.0 62.8 13.1 ST10E 166.496.5 66.5 10.4 ST30E 157.1 95.3 61.8 3.2 ST50E 157.0 112.1 89.0 49.2ST100E 172.5 118.0 92.2 65.0 control A 109.8 112.0 98.7 96.2 control B90.2 88.0 101.3 103.7 control A* 100.3 110.1 99.5 98.5 control B* 99.789.8 100.4 101.5 *analyzed week 2

The cell concentration and % cells in the S. cerevisiae flasks arepresented in Table 74. High cell counts were observed in all flasks;however, not all of the cells appear to be making ethanol.

TABLE 74 S cerevisiae Cell Counts and % Cells Cell Count % Cells Sample(cells × 10⁸/mL) (count/count control) *100 Number 24 hours 72 hours 24hours 72 hours P 0.62 0.96 97.7 139.0 P1E 0.35 1.18 54.1 170.9 P5E 1.131.93 177.3 279.5 P10E 0.59 1.42 91.8 205.6 P50E 0.32 1.40 49.4 202.8P100E 0.45 1.94 70.6 281.0 G 0.74 3.48 116.5 504.0 G1E 0.68 3.65 107.1528.6 G5E 0.62 3.87 96.5 560.5 G10E 0.70 2.73 109.5 395.4 G50E 0.46 2.1071.8 304.1 A 0.55 3.53 86.0 511.2 A1E 0.83 3.45 130.7 499.6 A5E 0.673.53 104.8 511.2 A10E 0.53 1.95 83.6 282.4 A50E 0.66 1.62 103.5 234.6 S0.44 1.11 69.5 160.8 S1E 0.44 1.10 68.2 159.3 S5E 0.23 0.99 36.5 143.4S10E 0.39 0.73 61.2 105.4 S30E 0.31 0.71 48.3 102.1 S50E* 0.44 0.90 86.5196.5 S100E 0.53 0.84 82.4 121.7 C 0.45 1.81 70.6 262.1 C1E 0.71 2.40110.6 347.6 C5E 0.53 2.33 83.6 337.4 C10E 0.77 1.55 120.0 224.5 C30E0.75 1.80 117.6 260.7 C50E 0.64 1.70 100.1 246.2 C100E 0.81 1.51 127.1218.7 ST 0.75 1.75 117.6 253.4 ST1E 0.57 1.36 89.4 197.0 ST5E 0.58 1.4990.7 215.8 ST10E 0.61 1.32 95.4 191.2 ST30E 0.59 0.60 91.8 86.9 ST50E0.59 1.30 91.8 188.3 ST100E 0.41 1.24 63.5 179.6 control A 0.81 0.79127.1 114.1 control B 0.47 0.59 72.9 85.9 control A* 0.66 0.42 131.291.7 control B* 0.35 0.50 69.0 108.1

The P. stipitis seed flask had an optical density (600 nm) of 5.01 and acell concentration of 3.30×10⁸ cells/mL. One mL of seed flask materialwas added to each of the test flasks. Therefore, the starting cellconcentration in each flask was 3.30×10⁶/mL. During the second week oftesting, the P. stipitis seed flask had an optical density (600 nm) of5.45 and a cell concentration of 3.83×10⁸ cells/mL. One mL of seed flaskmaterial was added to each of the test flasks. Therefore, the startingcell concentration in each flask was 3.83×10⁶/mL. The pH of the P.stipitis flasks at a sample time of 0 hours is presented in Table 75.The pH of the flask contents was within the optimal pH for P. stipitisgrowth (pH 4-7). No pH adjustment was required.

TABLE 75 pH of P. stipitis Flasks at Sample Time 0 Hours Sample NumberpH P 4.91 P1E 4.87 P5E 4.90 P10E 4.78 P50E 4.46 P100E 4.24 G 5.45 G1E5.43 G5E 5.48 G10E 5.32 G50E 4.99 A 5.69 A1E 5.66 A5E 5.60 A10E 5.58A50E 5.69 S 5.00 S1E 4.94 S5E 4.86 S10E 4.78 S30E 4.51 S50E 4.27 S100E4.08 C 5.36 C1E 5.30 C5E 5.29 C10E 5.06 C30E 4.89 C50E 4.70 C100E 4.59ST 4.93 ST1E 4.90 ST5E 4.81 ST10E 4.83 ST30E 4.91 ST50E 4.24 ST100E 4.07control A 4.93 control B 4.91

The ethanol concentration and performance in the P. stipitis flasks arepresented in Table 76 and 77. The highest ethanol concentrations werethe G and A series. Flasks C-30e, C-50e, and C-100e also contained highconcentrations of ethanol. The cell concentration and % cells in the P.stipitis flasks are presented in Table 78. Low cell concentrations wereobserved in the flasks with the S designations. Low cell counts werealso observed in flasks containing samples C, C1E, C5E, and C10E at the24 hour sample time.

TABLE 76 Ethanol concentration in P. stipitis flasks Sample EthanolConcentration (g/L) at the following times (hours) Number 0 6 12 24 3648 72 P 0.01 0.05 0.26 4.98 8.57 14.10 17.00 P1E 0.02 0.03 0.04 4.249.03 12.40 17.30 P5E 0.02 0.03 0.42 6.72 12.40 15.60 18.60 P10E 0.020.02 0.01 1.38 8.69 13.00 17.00 P50E 0.01 0.02 0.02 0.03 3.77 10.5016.90 P100E 0.02 0.03 0.02 3.75 10.50 15.60 18.80 G 0.02 0.08 0.20 10.8017.70 19.40 25.40 G1E 0.04 0.12 0.50 12.20 19.60 23.80 28.60 G5E 0.070.14 0.73 12.50 19.10 24.50 27.50 G10E 0.04 0.19 0.42 10.20 19.10 22.9028.20 G50E 0.05 0.22 0.25 8.73 18.40 22.20 28.00 A 0.13 0.28 0.82 16.1019.40 19.30 18.60 A1E 0.22 0.59 1.08 16.10 22.40 27.60 27.70 A5E 0.320.43 0.43 10.60 22.10 27.10 28.10 A10E 0.33 0.61 1.15 14.90 22.00 27.1027.90 A50E 0.30 0.10 0.47 13.40 20.20 24.80 27.10 S 0.01 0.01 0.26 3.687.50 10.20 13.30 S1E 0.02 0.02 0.22 4.98 9.22 11.60 14.20 S5E 0.02 0.020.19 4.25 8.50 11.70 14.70 S10E 0.03 0.02 0.17 2.98 8.87 11.90 14.70S30E 0.08 0.05 0.03 2.96 8.73 12.60 16.50 S50E 0.08 0.05 0.04 2.24 6.137.95 12.50 S100E 0.11 0.10 0.08 3.36 7.82 10.50 13.90 C* 0.02 0.03 0.050.23 1.66 2.68 6.57 C1E* 0.03 0.03 0.03 0.07 0.95 1.85 10.20 C5E* 0.030.02 0.04 0.05 0.37 1.59 4.80 C10E* 0.03 0.04 0.04 0.05 3.91 15.20 28.30C30E 0.01 0.03 0.60 12.30 21.20 26.00 27.20 C50E 0.02 0.02 0.45 12.3019.50 23.80 29.20 C100E 0.05 0.04 0.38 11.40 18.70 22.90 27.70 ST 0.030.03 0.37 6.69 10.70 13.50 10.90 ST1E 0.01 0.00 0.48 5.24 9.37 12.5015.70 ST5E 0.02 0.03 0.29 5.45 10.10 11.90 14.70 ST10E 0.02 0.02 0.425.60 9.44 12.20 14.90 ST30E 0.05 0.04 0.73 5.70 9.50 12.10 15.20 ST50E0.02 0.05 0.19 5.16 9.47 12.70 15.20 ST100E* 0.07 0.15 0.11 4.98 10.7015.40 18.80 control A 0.02 0.03 0.37 4.05 7.50 9.24 11.50 control B 0.020.02 0.30 4.22 7.44 9.44 11.50 control A* 0.02 0.05 0.69 4.86 8.69 11.1016.40 control B* 0.02 0.05 0.74 5.96 10.80 13.00 14.00 *analyzed week 2

TABLE 77 Performance in P. stipitis flasks Sample Performance (%) at thefollowing times (hours) Number 24 36 48 72 P 120.3 114.7 151.0 147.8 P1E102.4 120.9 132.8 150.4 P5E 162.3 166.0 167.0 161.7 P10E 33.3 116.3139.2 147.8 P50E 0.7 50.5 112.4 147.0 P100E 90.6 140.6 167.0 163.5 G260.9 236.9 207.7 220.9 G1E 294.7 262.4 254.8 248.7 G5E 301.9 255.7262.3 239.1 G10E 246.4 255.7 245.2 245.2 G50E 210.9 246.3 237.7 243.5 A388.9 259.7 206.6 161.7 A1E 388.9 299.9 295.5 240.9 A5E 256.0 295.9290.1 244.3 A10E 359.9 294.5 290.1 242.6 A50E 323.7 270.4 265.5 235.7 S88.9 100.4 109.2 115.7 S1E 120.3 123.4 124.2 123.5 S5E 102.7 113.8 125.3127.8 S10E 72.0 118.7 127.4 127.8 S30E 71.5 116.9 134.9 143.5 S50E 54.182.1 85.1 108.7 S100E 81.2 104.7 112.4 120.9 C* 4.2 17.0 22.2 43.2 C1E*1.4 9.7 15.4 67.1 C5E* 0.9 3.8 13.2 31.6 C10E* 0.9 40.1 126.1 246.1 C30E297.1 283.8 278.4 236.5 C50E 297.1 261.0 254.8 253.9 C100E 275.4 250.3245.2 240.9 ST 161.6 143.2 144.5 94.8 ST1E 126.6 125.4 133.8 136.5 ST5E131.6 135.2 127.4 127.8 ST10E 135.3 126.4 130.6 129.6 ST30E 137.7 127.2129.6 132.2 ST50E 124.6 126.8 136.0 132.2 ST100E* 120.3 109.7 127.8123.7 control A 97.8 100.4 98.9 100.0 control B 101.9 99.6 101.1 100.0control A* 89.8 89.1 92.1 107.9 control B* 110.2 110.8 107.9 92.1*analyzed in week 2

TABLE 78 P. stipitis Cell Counts and % Cells Cell Count % Cells Sample(cells × 10⁸/mL) (count/count control) *100 Number 24 hours 72 hours 24hours 72 hours P 2.78 11.00 80.6 148.0 P1E 2.10 7.20 60.9 96.9 P5E 2.939.68 84.9 130.3 P10E 1.42 7.73 41.2 104.0 P50E 0.33 8.63 9.6 116.2 P100E1.58 8.25 45.8 111.0 G 1.50 14.20 43.5 191.1 G1E 3.90 8.10 113.0 109.0G5E 2.93 6.45 84.9 86.8 G10E 4.35 13.30 126.1 179.0 G50E 3.75 11.60108.7 156.1 A 7.43 8.55 215.4 115.1 A1E 4.13 9.53 119.7 128.3 A5E 3.689.75 106.7 131.2 A10E 4.50 7.50 130.4 100.9 A50E 6.23 5.33 180.6 71.7 S3.53 5.55 102.3 74.7 S1E 3.00 3.30 87.0 44.4 S5E 3.68 3.00 106.7 40.4S10E 1.73 5.78 50.1 77.8 S30E 2.55 5.48 73.9 73.8 S50E 2.63 6.15 76.282.8 S100E 2.25 4.43 65.2 59.6 C* 0.00 0.26 0.00 7.2 C1E* 0.00 0.36 0.009.9 C5E* 0.00 0.08 0.00 2.1 C10E* 0.00 5.85 0.00 160.7 C30E 5.78 4.20167.5 56.5 C50E 3.40 7.35 98.6 98.9 C100E 1.98 6.60 57.4 88.8 ST 2.557.65 73.9 103.0 ST1E 2.00 8.70 58.0 117.1 ST5E 1.85 6.75 53.6 90.8 ST10E1.83 5.40 53.0 72.7 ST30E 2.78 6.15 80.6 82.8 ST50E 1.33 3.45 38.6 46.4ST100E* 4.35 3.83 59.8 105.2 control A 3.60 7.13 104.3 96.0 control B3.30 7.73 95.7 104.0 control A* 7.50 3.23 103.0 88.7 control B* 7.054.05 96.8 111.3 *analyzed week 2Cell Toxicity Results SummaryZymomonas mobilis

As shown in FIG. 7, elevated cell numbers (e.g., greater than thecontrol) were observed in samples containing P-132-10, G-132-10, andWS-132-10 at the 24 hour time point. Cell numbers in the presence of allother samples were comparable to the control. This observation indicatesthat the substrates were not toxic towards Z. mobilis for up to 24 hoursafter seeding.

At the 36 hour time point, a decrease in cell numbers (e.g., due to aloss of cells or cell death) was observed for all samples, including thecontrol. The greatest decrease in cell numbers was observed for thosesamples containing P-132-10, G-132-10. The likely cause of this effectis common to all samples, including the control. Thus, the cause of thiseffect is not the test substrates, as these vary in each sample, and arenot present in the control. Possible explanations for this observationinclude inappropriate culture conditions (e.g., temperature, mediacompositions), or ethanol concentrations in the sample.

As shown in FIG. 7, all cells produced comparable amounts of ethanol(e.g., 5-10 g/L) at each time point, irrespective of the substrate.Consistent with the cell number data presented in FIG. 8, ethanolconcentration in each sample peaked at the 24 hour time point. Incontrast to the cell number data, ethanol concentration did not decreaseat subsequent time points. This was expected as ethanol was not removedfrom the system. In addition, this data suggests that ethanol productionin these samples may have resulted from fermentation of glucose in theculture media. None of the substrates tested appeared to increaseethanol production.

Together, FIGS. 7 and 8 suggest that ethanol concentrations above about6 g/L may be toxic to Z. mobilis. This data is also presented as apercentage normalized against the control, as shown in FIG. 9.

Pichia stipitis

As shown in FIG. 10, cell numbers were comparable to the control.Furthermore, although slightly reduced cell numbers were present insamples containing G-132 and WS-132, reduced cell numbers were notobserved for G-132-10, G-132-100, A-132-10, or A-132-100. Thus, it isunlikely that substrates G or A are toxic. Rather, the reduced cellnumbers observed for G-132 and WS-132 are likely to have been caused byan experimental anomaly or by the presence of unprocessed substratesomehow impeding cell growth. Overall, this data suggests that glucosepresent in the control and experimental samples is likely to besufficient to promote optimal P. stipitis growth, and that the presenceof an additional substrate in the sample does not increase this growthrate. These results also suggest that none of the samples are toxic inP. stipitis.

As shown in FIG. 11, despite the similar cell numbers reported in FIG.11, greatly increased ethanol production was observed in all samplescontaining an experimental substrate. Ethanol concentrations increasedover time for each of the three time points tested. The highestconcentration of ethanol was observed for A-132-10 at the 48 hour timepoint (e.g., approximately 26.0 g/L). By comparing the substrateconcentrations with the highest levels of ethanol production with thecell number data presented in FIG. 11, it can be seen that P. stipitisdo not appear to be sensitive to increasing ethanol concentrations.Furthermore, ethanol production does not appear to be related to cellnumber, but rather appears to be related to the type of substratepresent in the sample.

Together, the results presented in FIG. 10 and FIG. 11 suggest that theexperimental substrates do not promote increased P. stipitis growth,however, they greatly increase the amount of ethanol produced by thiscell type. This data is also presented as a percentage normalizedagainst the control, as shown in FIG. 12.

Saccharomyces cerevisiae

As shown in FIG. 13, G-132-100, A-132, A-132-10, A-132-100, and WS-132promoted slightly elevated cell numbers compared to the control. Nosignificant reductions in cell number were observed for any sample.These results suggest that none of the samples are toxic in S.cerevisiae.

As shown in FIG. 14, increased ethanol production was observed in cellstreated with each cell type compared to the control. Comparison of thosesamples containing the highest amount of ethanol with the cell numberdata presented in FIG. 13 suggests that ethanol concentrations in excessof 5 g/L may have had an adverse effect on cell numbers. However, thisobservation is not the case for all samples.

This data is also presented as a percentage normalized against thecontrol, as shown in FIG. 15.

In conclusion, none of the samples tested appeared to be toxic in Z.mobilis, P. stipitis, or S. cerevisiae. Furthermore, P. stipitisappeared to be the most efficient of the three cell types for producingethanol from the experimental substrates tested.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

For example, the fibers can be in any desired form, and can have avariety of different morphologies. Generally, it is desirable that thecellulosic material have a high surface area. In some cases, the fibersmay be incorporated into single or multi-layer sheets, e.g., the fibersmay be part of a HEPA filter or the like. The sheet material can have asurface area of, for example, from about 1 to 500 m²/g. The fibrousmaterial can be overlaid, e.g., meltblown, folded, in the form of ascreen or mesh, or provided in other geometries. The fibers may beextruded or coextruded.

The fibers may have any desired particle size, from nano-scale, e.g.,less than about 1000 nm, e.g., less than 500 nm, 250 nm, 100 nm, 50 nm,25 nm, or even less than 1 nm, to large particle sizes, e.g., greaterthan 100 microns, 200 microns, 500 microns or even 1000 microns, oragglomerates of particles.

While biomass substrates have been discussed herein, such substrates canbe used in combination with other substrates, for example the inorganicand synthetic substrates disclosed in U.S. Provisional Application No.61/252,300, filed Oct. 16, 2009, the full disclosure of which isincorporated herein by reference.

The fibers or a fibrous material containing the fibers may be pretreatedwith a microorganism and/or enzyme, and/or the fibers or fibrousmaterial can be contacted with a microoganism and/or enzyme during abioprocess such as saccharification or fermentation.

As discussed above, enzymes can be immobilized on the fibers, instead ofor in addition to microorganisms.

Enzymes and biomass-destroying organisms that break down biomass, suchas the cellulose and/or the lignin portions of the biomass, contain ormanufacture various cellulolytic enzymes (cellulases), ligninases orvarious small molecule biomass-destroying metabolites. These enzymes maybe a complex of enzymes that act synergistically to degrade crystallinecellulose or the lignin portions of biomass. Examples of cellulolyticenzymes include: endoglucanases, cellobiohydrolases, and cellobiases(β-glucosidases). During saccharification, a cellulosic substrate isinitially hydrolyzed by endoglucanases at random locations producingoligomeric intermediates. These intermediates are then substrates forexo-splitting glucanases such as cellobiohydrolase to produce cellobiosefrom the ends of the cellulose polymer. Cellobiose is a water-soluble1,4-linked dimer of glucose. Finally cellobiase cleaves cellobiose toyield glucose.

A cellulase is capable of degrading biomass and may be of fungal orbacterial origin. Suitable enzymes include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,Chrysosporium and Trichoderma, and include species of Humicola,Coprinus, Thielavia, Fusarium, Myceliophthora, Acremonium,Cephalosporium, Scytalidium, Penicillium or Aspergillus (see, e.g., EP458162), especially those produced by a strain selected from the speciesHumicola insolens (reclassified as Scytalidium thermophilum, see, e.g.,U.S. Pat. No. 4,435,307), Coprinus cinereus, Fusarium oxysporum,Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris,Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremoniumbrachypenium, Acremonium dichromosporum, Acremonium obclavatum,Acremonium pinkertoniae, Acremonium roseogriseum, Acremoniumincoloratum, and Acremonium furatum; preferably from the speciesHumicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthorathermophila CBS 117.65, Cephalosporium sp. RYM-202, Acremonium sp. CBS478.94, Acremonium sp. CBS 265.95, Acremonium persicinum CBS 169.65,Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.71,Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae CBS157.70, Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS146.62, and Acremonium furatum CBS 299.70H. Cellulolytic enzymes mayalso be obtained from Chrysosporium, preferably a strain ofChrysosporium lucknowense. Additionally, Trichoderma (particularlyTrichoderma viride, Trichoderma reesei, and Trichoderma koningii),alkalophilic Bacillus (see, for example, U.S. Pat. No. 3,844,890 and EP458162), and Streptomyces (see, e.g., EP 458162) may be used.

Suitable cellobiases include a cellobiase from Aspergillus niger soldunder the tradename NOVOZYME 188™.

Enzyme complexes may be utilized, such as those available from Genencorunder the tradename ACCELLERASE®, for example, Accellerase® 1500 enzymecomplex. Accellerase® 1500 enzyme complex contains multiple enzymeactivities, mainly exoglucanase, endoglucanase (2200-2800 CMC U/g),hemi-cellulase, and beta-glucosidase (525-775 pNPG U/g), and has a pH of4.6 to 5.0. The endoglucanase activity of the enzyme complex isexpressed in carboxymethylcellulose activity units (CMC U), while thebeta-glucosidase activity is reported in pNP-glucoside activity units(pNPG U). In one embodiment, a blend of Accellerase® 1500 enzyme complexand NOVOZYME™ 188 cellobiase is used.

Accordingly, other embodiments are within the scope of the followingclaims.

The invention claimed is:
 1. A fermentation system comprising: afunctionalizing module configured to produce a functionalized biomass, asubstrate-forming module configured to immobilize a microorganism on thefunctionalized biomass, producing a substrate in the form of a fibroussheet having the microorganism immobilized thereon by ionic bonding ofcomplementary charged groups on the functionalized biomass and themicroorganism, a fermentation tank, a substrate delivery systemconfigured to deliver the substrate to the fermentation tank, and arecovery module configured to recover the substrate after a fermentationprocess, wherein the functionalizing module includes a particle beamaccelerator for irradiating a biomass feedstock and forming functionalgroups thereupon, and the substrate delivery system is configured toalso deliver a low molecular weight sugar dissolved in a solution to thefermentation tank.
 2. The fermentation system of claim 1 wherein thesheet is a multilayer sheet.
 3. The fermentation system of claim 1wherein the sheet is overlaid, folded, or in the form of a screen ormesh.
 4. The fermentation system of claim 1 wherein the biomassfeedstock comprises internal fibers that has been sheared to an extentthat its internal fibers are substantially exposed.
 5. The fermentationsystem of claim 4 wherein the biomass fibers have a porosity greaterthan 70%.
 6. The fermentation system of claim 4 wherein the biomassfibers are extruded or coextruded.
 7. The fermentation system of claim 1further comprising a module for mechanically treating the biomassfeedstock.
 8. The fermentation system of claim 7 wherein the module formechanically treating is designed to change the morphology of thebiomass feedstock, wherein the morphology that is changed is selectedfrom the group consisting of surface area, porosity, bulk density andmechanically treated fiber length to width ratio.
 9. The fermentationsystem of claim 8 wherein the mechanically treated fibers have anaverage length of 0.5 mm to 2.5 mm and the length-to-diameter ratio isgreater than 8/1.
 10. The fermentation system of claim 1 wherein thefunctionalization module can deliver a radiation dose of between 1 and100 Mrad.
 11. The fermentation system of claim 1 wherein the systemincludes one or more holding tanks, stripping columns, distillationunits, mixers and drying units.
 12. The fermentation system of claim 1wherein the functionalization module is located at a different site fromthe substrate delivery system and fermentation tank, and the systemincludes a method of transportation between the different sites selectedfrom a truck, a boat, a plane or a train.
 13. The fermentation system ofclaim 1 wherein the recovery module is configured to recover thesubstrate with the microorganism in a condition that will allow thesubstrate to be reused for more than one fermentation.
 14. Thefermentation system of claim 1, wherein the particle beam acceleratordelivers particles heavier than electrons.
 15. The fermentation systemof claim 14, wherein the particles heavier than electrons are nitrogenatoms or oxygen atoms.
 16. The fermentation system of claim 14, whereinthe particles heavier than electrons are phosphorous atoms.
 17. Thefermentation system of claim 14, wherein the particles heavier thanelectrons are sulfur atoms.
 18. The fermentation system of claim 14,wherein the particles heavier than electrons are selected from the groupconsisting of protons, helium nuclei, argon ions, silicon ions, neonions, carbon ions, phosphorus ions, oxygen ions and nitrogen ions.